The Nonradioactive Labelling of
Biologically Important Molecules
A Thesis Submitted Towards the Degree of
Doctor of Philosophy
by
Jeffrey Gore B.Sc.(Hons)
Department of Chemisury
The University of Adelaide
March 1996.
Contents
Acknowledgements
1
Statement
11
Abstract
111
Abbreviations
1V
Chapter 1-
Introduction
1.1.
Radioisotopic Labelling
1
1.2.
Nonradioisotopic Labelling of Biomolecules
2
1.3.
Characteristics and Detection of Labels
3
t.4.
Attachment of Nonradioactive Labels to Biomolecules
10
1.5.
Palladium Catalysed Couplings of Nonradioactive Labels
and Biomolecules
L9
t.6
Aim of Project
Chapter 2-
Synthesis of Label-spacer Molecules
2.1
Synthesis of Linker-spacer Molecules
27
2.2
Synthesis of Fluorescent Label-spacer Molecule Adducts
33
2.3
Synthesis of Time Resolved Fluorescence Label-spacer Adducts
42
2.4
Synthesis of Biotin Label
51
Chapter 3-
Preparation and Coupling Reactions of Biomolecules
3.1
Preparation and Coupling Reactions of Aminoacid Derivatives
52
3.2
Preparation and Coupling Reactions of Nucleoside Derivatives
66
3.3
Preparation and Coupling Reactions of Steroid Derivatives
72
22
Chapter
4-
Gelation of Organic Solvents by Biotin Amides and Esters
75
Chapter
5-
Summary
86
Experimental
87
References
t45
1
Acknowledgements
I would like to thank: my supervisor, Dr. Geoff Crisp, for his
assistance and
friendship throughout the years of this work; members of the Crisp research group,
particularly Markus Gerbauer and Tim Bubner for new ways and means; past and present lab
members for making the time pass quickly; the academic staff of the Department, who have
always been approachable about any subject; Daniela Caiazzafor proof reading this thesis;
and
finally, the technical staff who keep the department running. Also, the (increasingly
fewer) taxpayers of Austalia must be acknowledged for their contribution via a Postgraduate
Research Award.
It is easy to lose sight of the realities of life during
such an artifrcial situation as
studying for a higher degree, and so to my spouse Jane and children Antonia, Peter, Max and
Madeleine, thanþou for your love, support, pationce and for remembering who I am.
11
Statement
This work contains no material which has been accepted for the award of any other
degree or diploma in any university or other tertiary institution and, to the best of my
knowledge and belief, contains no material previously published or written by another person,
except where due reference has been made in the text.
I give consent to this copy of my thesis, when deposited in the University Library,
being available for loan and photocopying.
Signed:
Date:
:il.b. lE.rc.
111
Abstract
Radioisotopes, due to their radiation hazard and inherent instability, are being
replaced as reporters in biochemical systems by molecules which have luminescent or other
properties. The reporter molecules are covalently bound to the biomolecule of interest
generally via a nucleophilic/electrophilic interaction, hence the label is generally attached to a
hydrophilic site. The development and application of the palladium catalysed coupling
reaction between aryVvinyl halides/triflates and terminal alkynes to various biomolecule
derivatives suggested the possibility of using this methodolgy for the attachment of labels to
lipophilic positions in biomolecules. Hence fluorescent labels were synthesised from
acridone, pyrene, fluorescein and S-aminofluorescein by attachment to one terminus of a
hydrocarbon spacer arm, which had at the other terminus either an alkyne or a functional
group capable of being converted to an alkyne
.
Also synthesised similarly were
a
rris-phenanthrolineruthenium(Il) complex for use as a time resolved fluorescence reporter,
and a biotin label for use in the avidin/strepavidin reporting system. Suitable biomolecule
derivatives of amino acids (4-iodophenylalanine, 3-iodotyrosine, 5-O-triflyltryptophan and
4-O-ttflyltyrosine, propargyl glycine), nucleosides (5-iododeoxyuridine, 8-bromoadenosine,
8-bromoguanosine) and steroids (esuone and epiandrosterone triflate derivatives) were
prepared and labelling attempted with the above alkynes. It was found that palladium
catalysed cross coupling was an efficient and mild method for the introduction of the labels to
the biomolecules (except for uyptophan).
Unexpectedly, it was found that the biotin label 107 gelled some low polarity organic
solvents at low concentrations. Concentration dependent
tH NMR spectroscopy of the gel in
2[H]r-toluene were
CDCI3 and variable temperature studies of the gels in CDCI3 and
performed. In an attempt to define the scope of gelation by biotin compounds,
a series
saturated alkyl biotin esters and amides with varying chain lengths from n-propyl to
n-hexadecyl were synthesised and tested for gelation in many solvents. Gelation was
observed in hexane and paraffin oil for some derivatives.
of
1V
Abbreviations
18C-6
18-Crown-6
dansyl
5
DCC
dicyclohexylcarbodümide
DMAP
4-dimethylaminopyridine
DMF
dimethylformamide
DMSO
dimethylsulphoxide
DNA
deoxyribonucleic acid
FABMS
fast atom bombardment mass spectrometry
HRP
horseradish peroxidase
LAH
lithium aluminium hydride
LSIMS
liquid secondary ion mass spectometry
MEK
methyl ethyl ketone
mesyl, Ms
methanesulphonyl
NHS
N-hydroxysuccinimide
PCC
pyridinium chlorochromate
PdCC
palladium catalysed coupling
RNA
ribonucleic acid
RT
room temperature
TBAF
tetrabutylammonium fl uoride
TBDMS
t-butyldimethylsilyl
TEMPO
2,2,6,6-tetamethyl-
TI{F
tetrahydrofuran
TLC
thin layer chromatography
TRF
time resolved fl uorescence
triflate
trifl uoromethane sulphonate
triflyl
trifl uoromethanesulphonyl
tosyl, Ts
p-toluenesulphonyl
-dimethylamino- 1 -naphthalenesulphonyl
1
-piperidinyloxy free radical
Chapter L. Introduction
1.1 Radioisotopic Labelling
The elucidation of biochemical processes is fundamental to an understanding of the
nature of
lifel. With the discovery of methods in the 1930s for the a¡:tifrcial
transmutation of
stable atoms into non-stable isotopes, and for the collection of naturally occuring low
abundance isotopes, the availability of labelled elements and compounds allowed researchers
to trace the fate of vital substances ín vívo, and hence deduce facts about the mechanisms
which had occurred. In 1960 Broda wrote in his monograph2:
The importance of ísotopíc methods ín bíology has frequently been compared with that
of the microscope. Just øs the invention of the microscope ín the l7th century advanced the
science of living tíssues by tremendous strides, andmade possíble the later discovery of cells
and microbes, so does the employment of isotopic methods put us in ø posítion to ínvestigate
the details of metabolism ín a reliable and highly sensitíve manner.
From the end of the Second V/orld War Broda had seen the use of isotopic methods increase
dramatically3 and answer decisively many of the unresolved problems in biological science.
For example, in classic experiments the use of differentially labelled bacteriophage (2P
incorporated into the DNA and
3sS
materiala, and the incorporation of
into the protein coat) confirmed that DNA was the genetic
t5N
into E. coli bacteria showed the semiconservative
nature of DNA replications, consistent with'Watson and Crick's hypothesif.
The use of radioisotopicatly labelled compounds for research in biochemistry
laboratories is well established?. Substrates labelled with radionuclides can be detected in
very low amounts, and when the radionuclide rcplaces an atom of the same element the
chemistry of the system is unchanged (assuming the radioisotope is not involved in the rate
determing step and the concentration of radioisotope is low enough to ignore radiationchemical effects). Detection of the emined radiation is easy given the correct
instmmentation, the literature for introducing radionuclides into compounds is extensive, and
a large range
of labelled compounds is commercially available. A survey of commonly used
isotopes and relevant data is shown in Table 1. However, there are many disadvantages
of
Introduction
2
radionuclide labelling. The labelled compounds are a radiation hazard, requiring special
storage, handling and disposal facilities. Compounds labelled with short half-life isotopes
(".g."P, tãI¡ have a limited life before activity
levels drop below useful detection limits.
Laboratories and their workers which deal in radioisotopes are required to be government
approved. Quantitation of radioactivity levels in experiments may require
a
lengthy counúng
period. Although research laboratories will continue to use radioisotopes when necessary, the
disadvantages have spurred development of methods for the detection of labelled molecules
by nonradioactive means, especially for use in diagnostic labo¡atoriess where a longér shelf
life and shorter detecúon times a¡e distinct
advantages.
Table 1. Commonly Used Radioisotopes in the Biological
toc
3H
T,,
12.3
yr
decay mode
p
external
shielding
none
toxicty
slight
(ref. 2)
t.2
5730
yr
B
Sciencese.
35s
t2P
t2sI
1311
87.4 days
14.3 days
60.0 days
8.04 days
p
p
v
F+v
perspex lcm perspex lcm perspex lcm lead 0.25mm lead 13mm
slight
moderate
moderate
high
high
Nonradioisotopic Labelling of Biomolecules
A nonradioactively labelled biomolecule
can be considered to consist of three main
parts; the biomolecule, a spacer-Iinker arm and the label (a diagrammatic representation is
shown in Figure 1). When designing a labelled biomolecule, the main criterion is that the
"interaction of interest" (e.g. the ability of a labelled molecule to be recognised at the binding
site on a receptor, or to be used as a substrate by an enzyme) should not be significantly
affected. Most biomolecules have many potential points of attachment for the spacer or
spacer-label adduct;
a
judicious choice allows one to minimise the disruption to the
biomolecule's normal interactions. Secondary considerations, such as the type of label and
detection system to be used, and the relative polarity and solubility of the labelled
biomolecule compared to the unlabelled are also important, as the physicochemical properties
should not be changed significantly.
Introduction
bbmobcub
Iabel
spacer
Figure
3
1.
The spacer arm provides the reactive groups (as detailed below) for covalently linking
the label and biomolecule, and a means of preventing, or at least minimising, the steric
interaction between the label and the substrate. Depending on the label and biomolecule to be
joined, the reactive groups may be the same or different; in the latter case chemical
compatibility is necessary. The length of the spacer may vary from zero to sixteen or moreto,
the choice in some cases being a compromise between the requirements of the biomolecule
and the
label. For example, biotinylated nucleotides with shorter spacer arms are
incorporated more readily by DNA polymerases into DNA than those with longer spacer
arms, however detection of the biotin by avidin/strepavidin conjugates increases with
increasing spacer arm lengthlr. Functionalities such as amide and ester linkages are
commonly incorporated to modify characteristics (such as the improvement of water
solubilitys) of the labelled biomolecule. The order of attachment of biomolecule and label is
normally determined by synthetic considerations and the use of the labelled molecule.
1.3 Characteristics and Detection of Labels
Labelled biomolecules may be detected via direct or indirect systems (Figure 2).
Direct detection is used when the biomolecule is covalently bound to the reporter; the most
common reporter grcups are fluorescent dyes or marker enzymes (which catalyse the
formation of the detection signal) coupled with a luminescent compound. These systems
have the advantage that detection occurs in a single step, however an individual labelled
biomolecule must be synthesised for each application, and detection systems must be
available for all reporters. Indirect detection occurs when the reporter group is not linked
covalently to the biomolecule, but indirectly through
a
non-covalent intetaction between a
modification of the probe, and a molecule which binds specifically to that modif,rcation. The
most common modification group is biotin, and it is commonly detected via binding to avidin
Introduction 4
or strepavidin which is covalently bound to
a reporter
enzyme. A disadvantage is that
detection requires an extra step to allow the specific non-covalent interaction between the
modification group and the specific binding partner to occur, however the reporter group and
the detection system may be the same in all biomolecular systems. Hence indirect systems
tend to be used in basic research, and also in applied areas such as genetic engineering where
the detection of different target molecules is necessary.
Direct
systems
Indirect systems
Reporær goup
Bindinggor¡p
Modification goup
Probe
Target mobcub
Figure
2.12
Generally either an enzyme (commonly alkaline phosphatase (AP) or horseradish
peroxidase GIRP)) or a fluorescent repofter is covalently bound to avidin for detection
purposes. The enzyme catalyses a chemical reaction (which depends on the type of enzyme)
which generally involves the conversion of
a colourless
compound into a strongly coloured
derivative, which is the detection signal. For example, 3,3',5,5'-tetramethylbenzidine
I
in the
presence of HRP and hydrogen peroxide is oxidised to coloured 2 (Scheme 1), thus allowing
localisation of the labelled biomolecule.
NHz
HzN
IlC
CH¡
1:
?r,-",
NH
HN
->
CH¡
H3
22 Ìu
= 285 nm
Scheme
1. HRP,ItrOr.
"*=
450 nm
Introduction
5
Luminescencet', the emission of photons from electronically excited states, is the
property most commonly used for direct detection, and sometimes (vlø conjugation of a
luminophore to the binding group) in indirect detection. Molecules in electronically excited
states may be generated either by irradiation with photons of the required energy, or as part
a chemical reaction (chemílurnínescence). The absorption
approximately
first
(S1)
10-15 second, and
of
of a photon occurs in
gives a molecule generally in a vibrational level (v") of the
or second (Sr) electronic states. Molecules in condensed phases usually relax rapidly
(approximately
10-12
second) to the lowest vibrational level vo of S, (ínternøl conversion), and
many processes are now available for relaxation to the ground stateSo (Figure 3, Jablonski
diagram). Fluarescence occurs when emission of
a
photon from S, allows the molecule to
regain So; the transition is spin allowed and hence occurs quickly (the average lifetime of
fluorescent states are near
10-8
second). Intersystem crossíng to the triplet state T, may also
occur, but now the transition toSo is spin forbidden and emission of a photon occurs slowly
(triplet lifetimes generally are between
10-3
to
101 seconds); the
molecule exhibits
phosphorescence. Other processes, such as quenching, solvent relaxation, and reactions in the
excited state are also possible, and may decrease or negate the luminescent yield.
vn
52 u,
Vg
inæmalconversbn
vn
St vt
Vg
*
crossmg
absorption
To
ftrorescerrce
phosphorescerrce
vn
So vr
v0
Figure 3. Jablonski diagram
Introductíon
6
The most important optical properties of fluorescent moleculesra for use as reporters
are the wavelengths of maximum absorption and fluorescence emission, absorbance molar
extinction coeffrciente, and fluorescence quantum yield Q. The matrix may exhibit
background fluorescence at particular wavelengths and hence the reporter should be chosen
such that its fluorescence maximum is sufficiently different. Many specialised instruments
use a single fixed wavelength (usually an argon laser which emits at 488 nm), and
consequently the reporter must have significant absolption at this value. A large Stokes shift
(the separation of the absorbance and fluorescence wavelength maxima) is necessary to allow
the fluorescence signal to be isolated from backscattered excitation radiation. The
fluorescence intensity per reporter molecule, which is proportional to the product of e and
Q,
indicates the potential sensitivity available from a given molecule Other information apart
from spatial and temporal location, and concentration, may be obtained from fluorescent
molecules as fluorescence spectra and
Q are
affected by environmental factors including
solvent polarity, local environment (i.e. position in membranes, cells, proteins etc.),
proximity of quenching species and pH of aqueous media. Also, some fluorescent molecules
show enhancement of Q upon binding to a biomolecular target, as quenching by the aqueous
medium is diminished.
Another technique used is time resolved fluorescence (TRF), which is a system of
detection that has a potentially high degree of sensitivity. Certain europium (Itr) chelates
(e.g. 3) have been used as reporterst5 as they have a long fluorescent lifetime of 106 to 10r
second, which enables the differentiation of the reporter's fluorescence signals from any
background or native fluorescence. After short pulse excitation of the system (generally by a
laser) a time delay of 200 to 400ps occurs before the start of detection. This allows the
background fluorescence (with an average lifetime of 1t8 second) to decay to a negligible
level. As the longer lifetime fluorescent
species are
still emitting photons they are readily
detected. Pulsing can be repeated and the signal summed. However, the thermodynamic
stability of some europium complexes is low, which results in ligand dissociation at low
concentrations. Bannwarth et. al.r6 have used ruthenium (tr) complexes such
as
4 as reporters
attached to probes; they are thermodynamically very stable, chemically inert and are strongly
Introduction
7
fluorescent for a relatively long time. The detection limit for DNA probes labelled with4 is
t'P-labelled probes prepared by nick translation.
below 10 14 M, which is similar to
Eu+3
CO"-
("
T
N
N
tr
.2PFa
(cHt5cooH
NCS
3
4
Classes of molecules that are commonly used as fluorescent reporters and probes'o aro
shown below. Xanthene dyes (e.g. fluorescein 5, rhodamine B 6) generally have high
extinction coefficients and quantum yields, and are used mainly as reporter compounds.
Their characteristics include good water solubility, pH sensitivity and susceptibility to
photobleaching (reduction in fluorescence intensity due to chemical reaction of the
electronically excited species). Polycyclic aromatic hydrocarbo,ns(e.g. pyrene 7, anthracene
8) have
a
high extinction coefficient and quantum yield. They are used
as probes
in lipophilc
systems as the fluorescence spectrum is altered by enviromental factors, and hence
information about the the probe's local environment may be obtained. Also, they may be
used as indicators of complexationlT or structurett due to the formation of excimers (excited
state dimers), which are formed due to proximity of probe molecules and show a significant
shift in À-.* in the fluorescence spectrum. Aminosulphonyl derivatives of polycyclic aromatic
hydrocarbons (e.g. mansyl9 and dansyl 10 sulphonamide adducts) are formed by reaction of
the corresponding sulphonyl chloride with an amine. Although the extinction coefficient
Introduction
8
value (and hence the overall fluorescence output) is only moderate, the spectral characteristics
are environmentally sensitive, hence 9 and 10 may be used to probe the local environment
the
label.
of
Dansyl chloride is commonly used for the determination of the N-terminal residue
of amino acids, and to prepüe fluorescent derivatives of drugs, amino acids, oligonucleotides
and proteins. Acridine 11 derivatives, in particular acridine orange 13, have been used for
many years as fluorescent biological stainsle. Derivative 15 has been attached to
oligonucleotides to increase the stability of the duplex formed upon binding to a
complementary strand2o, as the acridine moiety inserts (intercalates) between the nucleobases.
Acridone 12 is easily alkylated on the nitrogen2r, and fluorescence is maintained. Recently
fluorophores based on the 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene moiety (14)
(BODIPYTM¡ have been inroduced, which a¡e claimed to have superior spectral
characteristics to existing dyes22.
H
+
NEtz
6
5
8
7
o
-o
o
9
10
Introduction
M%N
N
1l
N
F
I
I
H
H
12
13
+
9
CT
N
/B
N
F
15
L4
Labels are generally attached to spacer molecules via a nucleophilic/electrophilic
interaction, hence the label molecule may require modification (either pre- or postsynthetic)
to introduce a suitably reactive functionality. Common electrophilic groups included in labels
are isothiocyanates (Scheme 1a), activated esters (such as N-hydroxysuccinimide and
p-nitrophenol esters) (Scheme 1b) and sulphonyl chlorides (Scheme 1c), which react most
readily with amines and sulphydryls. The most common nucleophilic moiety introduced is an
amine, which generally is alkylated (Scheme
ld) or acylated
(Scheme 1e). Once the
label-spacer adduct is formed, covalent bonding to a biomolecule may occur by many
methods, which a¡e discussed in the next section.
Introductíon
10
S
a. label-N:C:S + Y-spacer
Y = NHz, SH
il
label-N -C-Y-spacer
l
H
-->
o
il
b. label-C-X + Y-spacer ---------r>
¡ = p- nitropb**y, N- hydrorysrrccinimllyl
o
il
c. label-S-Cl
il
o
+
d. Iabel-NH2 + X-spacer
X=I,Br
e
label-NH2
?
J-spacer
Y = NHz, SH
?
-spacer
Y = NHz, SH
labet-fr-Y-sPacar
o
->
taUet-f
H
->
+ X-C-spacer
-spacer
o
il
label-N-Q-spacer
I
¡
= p-
nitroph**y,
->
N- hydrorysuccinimlty[
Cl
H
Scheme 1. General methods of label-spacer attachment.
L.4. Attachment of Nonradioactive Labels to Biomolecules.
The nonradioactive labelling of naturally occurring proteins, peptides and nucleic
acids is well describeds'r1'14'1e'æ'ã. Labelling reactions occur mainly at discrete residues in the
biopolymer, and the chemistry of the interaction is described in the labelling of residues
section below. Natíve proteins andpeptides are generally labelled via a nucleophilic group
on the hydrophilic sidechain of an amino acid residue reacting with an elecnophilic group at
the terminus of the spacer in an alkylation or acylation reaction, although many other
procedures are used less commonlyæ. The most reactive nucleophile is the sulphydryl group
of cysteine, followed by the amino group of lysine; selectivity for the amino group of lysine
in the presence of
a free sulphydryl
is difflrcult to achieve. Serine and threonine are rarely
used for labelling as the nucleophilicity of the hydroxyl group is
low compared to sulphydryl
and amino groups (which react preferentially). Also, the substrates are generally in aqueous
solution, and reagents of sufficient reactivity are decomposed by the solvent. By appropriate
choice of functional group on the spacer and reaction conditions selectivity for most other
Introduction
11
reactive functional groups in the protein may be achieved. Synthetic peptides may be
labelled as previously, or may incorporate a modifîed residue which is either labelled or may
easily be attached to a label. RNA and DN,F are generally labelled via enzymatic
incorporation of a modified nucleotide triphosphate using nick translation or random primer
labelling; photochemical reaction with an azide compound or
a
photoreactive intercalating
compound (such as psoralen); or via chemical modification (e.9. bromide derivation with
NBS and subsequent reaction with an amine). Synthetíc olígonucleotides may incorporate a
modified phosphoramiditeã at the S'-terminus (or less commonly the 3'-terminus, the
intemucleotidic phosphate linkage or C2' of the sugar) which is attached to a label, or a
nucleotide which is either labelled or is modified so that it can be easily attached to a 1abe125.
Amino acids (as mentioned above) are generally labelled via a nucleophilic group on a
sidechain. A larger range of coupling reactions are available for single residues or small
peptides as they may be soluble in organic solvents, hence more reactive functional groups
(which are hydrolysed in aqueous solutions) are available on the spacer unit; also they may be
subjected to more forcing conditions than proteins, which may be denatured. The stability
of
the functional group formed upon reaction with the spacer needs to be considered with
respecr to the proposed use of the labelled residue. The sulphydryl moiety
of.
cysteine roacts
fastest with common electrophilic moieties. Reaction with cr-haloacetamides (Scheme 2a)
occurs via an S*2 displacement of the iodide leaving group, while reaction with N-maleimides
(Scheme 2b) is via a Michael addition. The most specific groups for sulphydryl moieties are
the mercurial compounds (Scheme2c), as the formation of a strong sulphur-mercury bond
results.
The nucleophilic e-amino moiety of lysine reacts with o-haloacetyl andN-maleimide
compounds similarly to cysteine, although at a slower rate (Schemes 2d and 2e). Reaction
also occurs with compounds with activated acid derivatives such as acid chlorides,
N-hydroxysuccinimde esters andp-nitrophenol esters (Scheme 2f) to give acyl derivatives.
Isocyanates (Scheme 2g), isothiocyanates (Scheme 2h) and sulphonyl chlorides (Scheme 2i)
also react with lysine to give N-alkylurea, N-alkylthiourea and N-sulphonamide derivatives
respectively. A free N-terminus of a small peptide reacts similarly.
Introduction
o
a.
il
C¡t-SH + I -CH2C-Q-sPacer #
b. Cfn-S
c.
+
-spacer
Cys-S
------>
Cys-S + Cl- + Hg-spacer +
d. Lys-NH, + I
-spacer
Cys-S-Hg-
spacer
o
o
il
..-_-+ Lys- N
-cH2c-o -spacer
I
H
H
e.
Lys-NH2
+
-spacer
f. Lys-NH2 +
Lyr-t
#
X = Cl, N-hydrox5nrrccinimlty[
(t
Lys-NH2
+ O:C:N-spacer
N-spacer
o
_-_>
X
+fÐ(
Lys_N
p- ninophenol
----------->
o
Lp- N
-N-spacer
I
I
H
H
S
tL
Lys-NH2
+ S:C:N-sPacer€
LYs- N
N-spacer
I
I
H
t.
Lys-NH2
+
Cl
Scheme
il
o
2.
12
spacer
-->
H
Lys- N
H
I
Reactions of cysteine (a-c) and lysine (d-i)
Introduction
13
Amino acids with an acidic side chain (glutamic andaspartic acíds) have been reacted
with carbodiimide derivatives and amines to form amides $cheme 3a), and also diazoacetate
ester and diazoacetamide derivatives $cheme 3b) resulting in esters. The C-terminus of a
small peptide may be tabelled similarly. Serine andthreonine may be labelled via the
hydroxy moiety with the more reactive acylating reagents such as acid chlorides to form
esters (Scheme
3c). Similarly, tyrosine may be labelled via the phenolic hydroxyl under
basic conditions (Scheme
as the resulting
3d). The other hydrophilic amino acids are generally not labelled,
functional groups do not have sufficient chemical stability.
carbodämile
+
a.
H2N-sPacer
------------>
N-spacer
R
I
H
+
+ N:N:C
b.R
o
o
+R
o
spacer
I
H
c. R-OH + Cl
d. Ar-O + CtScheme
3.
_____--> R
+
tu-o
-spacer
o
Reactions of carboxylic acid (a, b), hydroxyl (c) and tyrosine (d) sidechains
Nucleotide.f may have labels attached at the sugar (generally the 2'-hydroxy of
ribose26), or more commonly, at the nucleobase. Moieties involved in hydrogen bonding
which are used for molecular recognition should not be affected, and importantly, the
modified nucleotide must be a substrate for any enzymes used in the processes in which it
(the nucleotide) is involved. Attachment of the spacer or label-spacer adduct to the
nucleobase may be realised by a displacement reaction of a leaving group by a nucleophile
(generally an amine or sulphydryl group) of the spacer. Hence reaction of the good leaving
Introductíon
14
groups triazole 2027 and2,4,6-Íimethylphenol 2128 at C4 of both cytosine 16 and thymidine
derivatives 18 (Scheme 4) gave the labelled adducts
l7
and 19; other leaving groups have
also been used2e. Also, S-thymidine derivatives23 and 25 have been prepared by the
palladium mediated allylation3o of 22in poor yield, and by the palladium catalysed coupling
of protected propargylamine to 5-iodouridine derivative€' 24 (Schemes 5 and 6
respectively).
Adenosine derivatives 27 and29 have been synthesised in the 8-position via
nucleophilic displacement of bromide from 8-bromoadenosine26by amines32 and thiolate
groups33 (Scheme 7), and
2834
in the 6-position by reaction of amines with 6-iododeoxyadenosine
lScheme 8). Palladium catalysed coupling of an N-protected propargylamine to
3l and 33 (Schemes 9 and 10
7-deazadtdeoxypurines 30 and 32 gave the adductft
respectively), which were then deprotected and reacted with fluorescent labsls to give
compounds suitable for use as terminators in an automated Sanger dideoxynucleotide
sequencing protocole.
B2
p1
N
€
oÀ*
1
N
oÅ*
sugar
o
sugar
Rr=H
l8: Rr = CH3
17: Rr=H
19: Rt = CI{3
16:
Scheme
4:
RP
= spacer;
2l
20
X=20 or2l; Y = NH or S.
o
HÉI
HN
"L
N
l
sugaf
22
HN
N
N
H
K2PdCl4, pH 5 bttrer
I
sugar
23
Scheme 5.
l\
H
CF¡
Intoduction
HN
¿
H
HN
N
I
sugar
oÅ*
H
Pd(PPh3)4, CuI, Et N,
DMF
25
24
Scheme 6.
spacer-Y
N
(
Br -----+
N
N
(
-spacor
N
I
sugar
sugar
27
26
Scheme 7.
H-*-sPacer
N
)
(*
spacer-NH2
N
N
(
N
)
29
28
Scheme 8.
15
Introduction
16
o
H
N
CF¡
N
H
Pd@P\)a, CuI, EtrN, DMF
N
N
I
sugar
sugar
31
30
Scheme 9.
N
H
N
H
HN
H
Pd(PPh3)4, CuI, Et3N, DMF
HzN
HzN
sugar
32
33
Scheme 10.
Steroid derivatives are generally monomeric and so a wider range of chemistry may
be utilised in the labelling roactions, although labels ate commonly attached via interaction
with either hydroxyl $oups or carbonyl groups. Attachment of
hydroxyl group is common and simple (e.g. 34
)36,
a
fluorescent label via a
however a free hydroxyl group may be
necessary to maintain the physicochemical properties of the unlabelled molecule. Formation
of carbon-carbon bonds via a carbonyl group, although synthetically more involved, may
enable attachment of labels with minimal alteration of the physical properties. For example,
addition of phenyl lithium to 35 (Scheme 11) enabled formation of organometallic derivative
36, which was suitable for studying the hormone-receptor interaction for estradiol37. Also,
addition of a phosphonate ylid to 37 enabled the synthesis of a fluorescent cholesterol
analogue 38 (Scheme l2), suitable for use as a probe in membrane studies3s.
Introduction l1
ozN
NH-CIr2-(CIJz-OCHù2-CH2-NH
34
c(co)3
€
35
36
Scheme 10. (Ð PhLi,
-70'. (ii) ISiMer. (iiÐ Cr(CO)u.
------------>
RO
37
Scheme 11. (Ð (E)-@IO)rP(O)CTLCH=CHPh, ¿-BuLi,
38
TI#, -7S; (ii) 37; (iii) TBAF, THF.
Polysaccharides are commonly reacted with hydrazine and amine derivatives to
introduce labels3e. Addition of hydrazines to an aldehyde or ketone functionality in the
polysaccharide (which may be introduced by periodate oxidation of the commonly occurring
Introductíon
18
vicinal diols, if not originally present) gives a stable hydrazone adduct (Scheme 13).
However, addition of amines to the carbonyl groups is reversible and gives unstable Schiff
bases; subsequent reduction by NaBI{o or NaCNBH, results in stable amine conjugates
(Scheme 14).
N -NH-sPacer
H2N-NH-spacer
R
R'
R'
Scheme 13.
/.spacer
aBHa orNaCNBH3
H2N-sPacer
R
R
R
R
H
Scheme 14.
As has been shown, the formation of a covalent bond between a label and a
biomolecule generally requires a nucleophilic-electrophilic interaction. However, with the
application of modern transition-metal mediated chemistry such as the palladium-catalysed
cross coupling reaction of terminal alkynes with aryl or vinyl halides (as shown in Schemes
6,9 andlO), low polarity covalent
bonds may be formed readily between suitable carbon
atoms. Hence a label molecule may be attached to one terminus of a spacer which is
functionalised at the alternate terminus with a terminal alkyne (or a group which is converted
easily to a terminal alkyne), and then reacted under palladium catalysis with a bromo-, iodoor triflyl- arene or alkene functionality on a biomolecule (Scheme 15). The use of this type
of reaction for labelling biomolecules offers the primary advantage in that lipophilic sites on
biomolecules may be labelled, which is in general complementary to current methods. Thus
the aromatic sidechains of aminoacids such as phenylalanine, tyrosine and typtophan, the
nucleobases of adenosine, guanosine and deoxyuridine and derivatives of the steroids esffone
and epiandrosterone should be able to ¡eacted with label-spacer adducts, as suitable
halogenated or triflated derivatives are either commercially available or readily synthesised.
Introduction
19
Another advantage is that label-spacer adducts synthesised with a terminal alkyne
functionality may be coupled to any suitable biomolecule derivative in
a one-step
reaction;
it
is a general method. Also, a hydrocarbon spacer arm may be used to link the label and
biomolecule, which should be more resistant to enzymatic hydrolysis if used in vivo. than the
functional groups (e.g. esters and amides) which result from standard labelling rcactions.
label
+
-------.-->
spacer
Pd (caÐ
label
spacer
label
X
bionnlecub
spacer
bbmolecule
Scheme 14. X = I, Br, OTf
L.5. Paltadium Catalysed Couplings of Nonradioactive Labels
and Biomolecules
The use of palladium catalysis in the formation of carbon-carbon bonds in the
synthesis of complex organic molecules is well establishedo. The reactions are generally
high yielding, chemoselective, occur under mild conditions, are compatible with a wide range
of functional groups, and have been shown not to cause racemisation of amino acid
derivativesaT. A new bond is formed between an electrophilic
s/- or sp3-hybridised carbon
atom (a halo- or triflyt- derivative) and a nucleophilic (alkene or alkyne) carbon atom or
derivative, the major derivaives being cuproacetylidesal, organoboraneso2 lthe "Suzuki"
coupting) and organostannaneso' (the "Stille" coupling). The cross-coupling reaction between
acetylenes and aryl or vinyl halides under palladium catalysis was first reported by Heck4
and Cassaras independently
in 1975. A typical coupling between iodobenzene
and
phenylacetylene in EgN (Scheme 16, Conditions A) required elevated temperatures to
Introduction 20
proceed. Sonogashira's use of
a copper
iodide co-catalyst6 (e.g. Scheme 16, conditions B)
(which forms a nucleophilic copper acetylide) allowed this reaction to occur at room
temperature. Reaction of substrates which were not soluble in the amines initially used
as
solvents can take place in polar aprotic solvents such as DMF andDMSO. Many complex
organic molecules have been constructed using this methodology as a key synthetic stepot.
I
+
-¡>
Conditions
Conditions
A: Pd(OAc) (0.01 eq), PPh, (0.02 eq), Et N, 100", 1.5 hours,]3Vo.
B: Pd(PPhr)rClr(0.10 eq), CuI (0.05 eq), EqNH, RT, 6 hours, 857oScheme 16.
The catalytic cycle for the cross-coupling between aryVvinyl halides/triflates and
cuproacetylidesar is shown in Scheme 17. The palladium species may be introduced in an
oxidation state of zero (Pd(PPhr)/ or two (e.g. Pd(PPh3)2Ct). tn both cases the active
catalyst is the 14 electron, coordinately unsaturated, catalytic species
is formed by either the dissociation of two ligands from
prll,
PdI, (l- = PPh¡) which
upon dissolution in the reaction
solvenr, or by the reduction of PdtrLrClrviahomocoupling of the alkyne. Oxidative addition
of the electrophilic carbon occurs most quickly for iodides; bromides and triflates add at a
similar rate and chlorides are the slowest. Transmetallation of the alkynylcuprate (generated
in catalytic quantities from the reaction between CuI, EgN and alkyne) with the catalytic
species and reductive elimination of the coupled product regenerates the catalyst.
Introduction 2I
PdL¿
Ar:R
1I
Ar-X
Pdlz
redwtive eliminatbn
oxidative additbn
Ar
I
Pd
L
I
Ar
X
-L
L
tansnptalhtbn
+ Et N +
I
I
R-:Cu
CUX
R-H
Pd-L
R-:-Cu
CuI +
+ Et NH+
-I
Scheme 17.
A potential problem of the reaction is the production of the homocoupled alkyne in
preference to the cross-coupled product. Recovery of the homocoupled alkyne in a
significant amount has been reported when the oxidative addition of the aryl halide species is
slowas (generally due to the use
piperidineae or pyrrolidinesO at
of an aryl bromide or electron rich aryl iodide). Reaction in
reflux gives
a
higher yietd of cross coupled product, and hence
less homocoupled alkyne, however this approach is limited by the stability of the substrate
under the reaction
conditions. Another problem is chelation of the catalytic metal ions to
the substrate or product, or both; a sluggish reaction may result due to the reduced availability
of the catalysfr. Treatment of a solution of
with
tlS
a crude
purine nucleoside cross-coupled product
gas to precipitate the chelated metals as metal sulphides was necessary for isolation
of the desired products2. These disadvantages
are
readily overcome by suitable choices of
Introductíon 22
reaction conditions and/or substrate protection, and purification methods. Hence the
palladium catalysed cross-coupling of aryl or vinyl halides or triflates with terminal alkynes
should provide an efficient method of attaching a reporter compound to a suitable
biomolecule derivative.
t.6. Aim of Project
The aim of this project is to ascertain if the palladium catalysed cross-coupling
reaction is a suitable method for the introduction of label-spacer adducts, which are
functionalised with a terminal alkyne group, to iodo, bromo and triflyl derivatives of
biomolecules. The synthetic work consists of three main pafts; (i) synthesis of spacer
molecules and label-spacer adducts, (ii) synthesis of halogenated or triflated biomolecule
derivatives, and (iii) reaction of biomolecule derivatives and label-spacer adducts under PdCC
conditions to give the labelled biomoleclues.
Undec-lO-enoic acid was selected as a suitable stafting material for spacer molecules
(which need to be cr,r¡-difunctional hydrocarbon chains) as it was inexpensive, readily
available and of medium length. Interconversion of the functional groups to those suitable
for attachment to both the label and the biomolecule (which requires an alkyne or a group
easily converted to one) should be readily accomplished by standard chemistry. The
fluorescent labels selected for use in this study were fluorescein 5, pyrene 7, dansyl
sulphonamide derivative 10 and acrido ne 12. The complex tris(phenanthroline)ruthenium
(II) hexafluorophosphate 39 was selected as a model tíme resolvedflu.orescencereporler in
place of the bathophenanthroline ruthenium complex 27 as the ligand 40 is less expensive,
bromo derivatives of 1,l0-phenanthroline40 are easily synthesised from literature
procedures, and the coupling ability under palladium catalysis of both complexes should be
similar. Also 1,lO-phenanthroline derivatives
and complexes have many other interesting
chemical and physical properties which have been intensively studied3, and the methodology
developed may have applications in those areas. Finally, abiotin 41 label was tested for
coupling under PdCC conditions.
Introduction 23
H
H
H
s
N
.2PF6
o
4l
40
39
Synthesis of the label-spacer adducts involving the aromatic moities 7 and40 should
occur via P{CC of halogenated derivatives with an alþnyl spacer $cheme 18). After
coupling, the internal alkyne may be either hydrogenated to the saturated analogue, or
leftín
sita, which offers labels with possibly different specml characteristics. Finally, functional
group interconversion (FGD to the terminal
alþne gives the labels42 and43 which should
be suitable for coupling.
hbel
X+
spacer
.#
(Ð
(ä), (uD
hbel
spacer
(trÐ
label
42
Scheme 18. X = I,
spacer
Iabel
spacer
43
Br. (i) PdCC. (iÐ tHl. (iii) FGI
Synthesis of the other labels involves elecrophilic-nucleophilic interaction between
suitable functionalities of the label and spacer molecules; attack on an electrophilic alkynyl
spacer for the labels (acridone, fluorescein) which may possess a suitable nucleophilic site
such as an anionic heteroatom (Scheme 19) to give 44, or attack by a nucleophilic spacer on
Introductíon 24
an electrophilic label (dansyl chloride, biotin NHS ester) (Scheme 20) to give 45.
Iabel
Y+X
Y
Iabel
spacef
->
Scheme 19.
Iabel
X+Y
44
Y = ArO-, N-; X = I.
spacer
label
spacer
-l>
Scheme 20.
spacer
45
Y = NFL; X = Cl, NHS.
The biomolecules selected for labelling are halogenated or hydroxy derivatives (for
conversion to triflates using standard methodologfo) of amino acids and nucleosides which
are commercially available. Functional groups such as the hydroxyls of the sugars
nucleosides and the carboxylate and amino gÌoups of amino acids
will
of
be protected; although
not necessary for the PdCC reaction, protection will aid purification by chromatography and
cha¡acterisation of the labelled biomolecule. The halogenated amino acids selected to be
trialled were 4-iodophenylalanine 46 and 3-iodotyrosine 47; tyrosine 48 and
5-hydroxytryptophan 49 were to be converted to the respective triflates before labelling was
attempted. Also, reactions of protected propargyl glycine 50 (which is readily resolved into
both enantiomers, and has been shown to couple with aromatic and vinyl halides and
triflatesaT)
with l-halopyrenes to give a labelled glycine derivative will
be studied.
Halogenated nucleosides are readily available, and those selected for trial were
5-iododeoxyuridine 51, 8-bromoadenosine 52 and 8-bromoguanosine 53. Finally, as steroidal
triflate derivatives have been shown to couple readily to terminal
alkynesa855, the estrone
triflate derivative 54 and epiandrosterone triflate derivative 55 were selected for trial in
labelling reactions.
Introduction
I
H'o
*HrN
H
H,,
H,o
+HrN
coz-
25
*H¡N
coz-
Coz48
47
46
H
N
I
H'.
+HrN
AcN
coz-
H
50
49
HN
I
CO2Er
I
OH
N
ll-
H
Br
N
Br
N
HzN
HO
HO
HO
53
52
51
OH
Ac
TO
MeO
55
54
If
these biomolecules are successfully labelled using PdCC, the number of possible
labelling sites will be increased, in
a method complementary to
existing protocols. There are,
however, other advantages. The conformational change difference between labelled and
unlabelled potypeptides may be reduced as hydrogen bonding interactions of the sidechains
are not disrupted by heteroatoms being used for attachment of the
label. Also, the charge
Introduction 26
and,/or the pKa of the heteroatom, and hence the polypeptide, is affected by the covalent
bonding of the label. Both these problems may be overcome by attachment of the label to the
sidechain of a lipophilic residue. The use of a hydrocarbon spacer may offer advantages
when synthesising labelled biomolecules for use in lipophilic systems. Compared tQ the
labelling reactions for steroids (see chapter 1.3,[-abelling of Bíomolecules), use of this
methodology does not rcqufue the presence of heteroatoms for attachment (c.f.19) and so the
adduct should be less polar. Although some nucleosides have used PdCC for the introduction
of spacer molecules, labelling of nucleosides via this methodology should be simpler
as the
spacer-label adduct is intr. oduced in one step, compared to the three steps required for
coupling of the protected spacer, deprotection and coupling of the label as shown in Schemes
5, 8 and 9.
Successful exploitation of palladium catalysed coupling methodology would allow the
facile preparation of a large range of tabelled biomolecules, with the incorporation of
a
hydrocarbon spacer arm which is attached to a lipophilic portion of the biomolecule, in high
yields. The chemoselectivity of the coupling process should allow different label-spacer
adducts to couple to different biomolecules, hence the synthesis of novel labelled
biomolecules should be possible. This methodology is, in general, complementary to existing
labelling protocols.
Chapt et
2. Synthesis of Label-spacer Molecules.
Chapter 2.L. Synthesis of Linker'spacer Molecules.
Linker-spacer molecules were required to be c,crfunctionalised alkyl chains with an
alkyne occupying one of the termini, so as to be attached to a halogenated or triflated label
derivative or the biomolecule. The other ærminus could be a functional group suitable for
either attachment to the label, or for conversion to an alkyne which is then attached to the
biomolecute. The choice depends on whether
a
purely hydrocarbon linker arm is required or
whether some functionality is permitted. When PdCC is necessary for attachment of the label
the functional group at the other terminus should be inert to the coupling reaction conditions,
and
if required
subsequent conversion to an alkyne should be
facile. When an electrophilic
label is required to be attached, nucleophilic attack by the spacer with an amine functionality
is preferred as the resultant functional group (e.g. an amide) generally is stable under
projected reaction conditions. Conversely, attack on the spacer by a nucleophilic label
requires an elecnophilic group such as an iodide. The chemisEy involved in the functional
group interconversions should not depend on the length of the alkyl chain, and hence be
applicable to any alkyl length chosen. Undec-lO-enoic acid 56 is an inexpensive, readily
available, cr,cudifunctionalised molecule of medium length and was selected to provide the
starting material for the spacer-linker molecules.
Conversion of 56 to undec-10-ynoic acid 57 was via a literature method6.
Bromination of 56 in CClo at 0o gave the vicinal dibromide, which was then subjected to
double dehydrohalogenation. Dehydrohalogenations to give terminal alkynes have been
achieved using many methodssT, most commonly with NaNI{, in liquid ammonia or with
aqueous
KOH. The basicity of NaNfl is strong enough to form the salt of the ærminal
alkyne (which is thermodynamically more stable than the internal isomet's), and hence upon
protonation in the workup the major product is the terminal alkyne. The alternative method
of KOH in
IIO
was chosen due to its ease of workup; the low literature yield (327o) is
acceptable due to the low cost of 56. Afterreaction of the vicinal dibromide with KOH for 8
hours (Scheme 2l) at 150', and isolation of the product by distillation and recrystallisation
Results and
Discussions
28
tH NMR analysis showed resonances
f¡om hexane, the product was rocovered in 307o yieLd.
at
ô 1.94 (r, lH, J
2.6Hz,HC{)
and 2.18 (dt, 2IJ, J 7.0, 2.6H2, CH2-C=) which were
consistent with 57; also observed was an unexpected resonance at õ 1.78 (t, J 2.4IJ2). A
13C
NMR spectrum of the product showed alkyne resonances at ô 68.0 (terminal) and 84.6
(internal) which were consistent with 57, and low intensity resonances at õ 74.5 andl9.I.
Isomerisation of a terminal alkyne to the thermodynamically more stable internal alkyness has
been shown to occur under basic conditionsse (presumably via an allene intermediate); here
the unexpected resonance at õ 1.78 was consistent with the terminal methyl groupse of the
9-alkyne isomer 58, and the
13C
resonances also consistent
with an internal alkyneo.
Integtation of the terminal methyl group resonance with reference to the methylene next to
the carboxyl group (with allowance for the contribution from 58) showed the ratio of 57 to 58
to be 90:10. Repeated fractional distillations through a 150mm Vigreaux column could not
increase the ratio and so modifications to the reaction conditions were made to reduce the
amount of 58.
o
il
56
+CH
--------->
58
57
(i) Br, CCl4,0'. (ii) KOH (aq), 150', 8 hours,307o
Scheme 21.
Reducing the temperature and,/or time of reaction should lead to an increase in the
ratio of the kinetic product 57 to thermodynamic product 58, albeit with
yield. Consequently repeating the reaction at 130' for
a
reduction in overall
8 hours gave a ratio of 98:21or 57 to
58, with a yield of 247o, and reaction at 120' for 6 hours gave only 57 in a yield of 187o.
After distillation of the product, the majority of reaction mixture remained as high boiling
point residue, which was presumably a mixture of the bromoalkenes which are intermediates
in the reaction. As the reaction was performed on a large (100g) scale from starting material,
the poor yield was acceptable, in terms of quantity of material obtained.
Results and Díscussions 29
Reduction of 57 to the alcohol 59 occured readily in very good yield using LAH in
refluxing Et2O (Scheme22). Synthesis was confirmed by IR (3400-3100 cm-l, O-H str., and
absence
of carbonyl
tH NMR (õ 3.62, t,2H, C[L-OH).
absorption at 1720 cm-t¡ and
:(CH2)sCO2H
:-(cHteoH
€
59
57
LAH, Et O, reflux overnight, 87 Vo.
Scheme 22.
Conversion of 59 to the iodide 62 was via the standard two step procedure; conversion
of the alcohol to
a good leaving group (ie the tosylate 60 or the mesylate 61) and subsequent
displacement with NaI in 2-butanone6r. Reaction of 59 with TsCl in pyridine (Scheme 23)
gave an excellent crude yield of the corresponding tosylate 60. Reaction of 59 with MsCl in
pyridine gave the expected mesylate 61 also in excellent yield (Scheme 24' Conditions A).
The method of choice however, was reaction of 59 with MsCl and EqN in CIICI, to give 61
(Scheme 24, Conditions B), then conversion to 62. Although the yield of crude mesylate was
slightly lower than tosylate, (88Vo to 937o), the advantages were that no pyridine was used,
and that the isolation procedure was easiest. Displacement of the tosylaæ or mesylate groups
(Scheme 25) occurred readily to give the iodide 62 as a colourless oil in good yield; synthesis
was conf,rmed by mass spectrometry (M. 278) and the distinctive high field chemical shift in
the
13C
NMR spectrum (ô 7.18) of the carbon attached to the iodide.
:(CHre-oH
59
----------->
TsCl (2 eq), pyridine,
ü
-
(CH2)s-ors
60
to RT overníght;94Vo.
Scheme 23.
Results and Díscussíons 30
___________>
59
Conditions
Conditions
:(cH2)e_oMs
61
A: MsCl (1 eq), pyridine,0o,4 hours;937o.
B: MsCl (1 eq), Et3N (1 eq), CFIC!, RT,24
hours, 887o.
Scheme 24.
€
:-(CHte-OR
60: R =OTs;697o
:(CHzb-I
61: R =OMs;757o
62
NaI (5 eq), 2-butanone, reflux 12 hours.
Scheme 25.
Preparation of aminoalkyne 66 was envisaged to occur by a standard Gabriel
synthesis62. Reaction of 62 with the sodium anion of phthalimide 63 in
DMF (Scheme 26,
Conditions A) gave 64 in apoor yield of 4I7o. TLC (20/80 EtOAc/hexanes) of the reaction
mixture after 3 hours stirring at 100' showed
new UV active spot at
&
a large amount
of phthalimide at \ 0.22, and a
0.54 corresponding to product; the spot corresponding to 62 was
absent. Synthesis was confirmed by mass spectrometry (M.297) and IR (1772 and 1710
cm-t, imide C=O stretch). The yield was increased to78Vo by the reaction between 62,63
and
ÇCO, in refluxing 2-butanone (Scheme 26, Conditions B). In an attempt to improve
yield
a phase transfer catalysed reaction63 was
performed, however reaction of potassium
phthalimide 65 and iodide 62 in toluene with PTC catalyst 18C-6 (Scheme 27) gave upon
workup only
a
poor yield of 407o.
-H
63
Conditions
Conditions
A:
B:
-(CHz)s
------->
64
(i) NaH (1.1 eq), DMF. (ii) Iodide 62, 100",3hovs,4LVo.
Iodide 62 (1.2 eq), KrCOt Q.Z eq), MEK, reflux 24hours,78%o.
Scheme 26.
the
Results and Discussions 3L
_+K
-(CH)n:
->
65
64
Iodide 62 (1.2 eq), 18C-6 (0.1 eq), toluene, 90", 8 hours, 407o.
Scheme 27.
Reaction of 64 with 3 equivalents of hydrazine hydrate in EIOH at toom temperature
and standard workup (Scheme 28) gave the amine 66 in an average yield of 647o. Synthesis
was confirmed by IR (3500-3100 cm-l N-H stretch) and mass spectrometry (M+H. 168).
Varying the amount of hydrazine (1, 2 and 5 equivalents) did not improve the yield, neither
did reaction at a higher temperature. As the amine is four synthetic steps from the acid 57
with an overall yield of 347o, the alternative path of conversion of 57 to the amide 68 and
reduction to 66 with LAH was explored.
-(CHts
-------->
64
(Ð
H2N-(CH2)e:
66
rLNI{Hr.rLO (3 eq), EIOH, RT, 48 hours. (iÐ HCI (aq). (iii) NaOH (aq).
Scheme 28.
Reaction of acid 57 with SOCI2 gave the acid chloride 67 (Scheme 29 (Ð) in good
yield. The IR spectrum of 67 showed the absence of the O-H stretch between 3350
and 2500
cm-l, and a strong absorption at 1796 cm-l consistent with the carbonyl group of an acid
chloride. The tH NMR chemical shift of the methylene next to the acyl chloride was ô 2.88,
compared with ð 2.35 in 57. Addition of 67 to a cold (-15') saturated solution of ammonia
Results and Discussions 32
(Scheme 29 (iÐ) precipitated the amide 68 in very good yield. The IR spectrum showed
distinctive primary amide N-H stretch absorptions at 3356 and 3184 cm-r, and strong
absorptions at1662 (C=O str.) and 1632 cm-t
(Nf!
def.).
o
---_.-->
57
(i)
o
+
67
SOC12,
reflux, 60 minutes (ii) conc. aq. NHr, -15";84Vo-
68
Scheme 29.
The amide 68 was only partially soluble in EqO, hence it was placed in the thimble of
a Soxhlet extraction appamtus
for reduction to the amine 66 by LAH in EqO (Scheme 30).
After reflux for 36 hours the amide had dissolved and the amine was recovered in very good
yield; the IR spectrum showed a broad absorption between 3500 and 3100 cm-l (N-H str.) and
the
lH NMR
a resonance at ô
2.60
(t,zIJ,I
6.6}J:2) consistent with
yield for the two-step synthesis of 66 from the acid 57 was 737o,
CIlrNtl.
a marked
The overall
improvement
compared with the previous four-step synthesis of 34Vo.
o
68
__>
:(cHreNH2
LAH (1 eq), EtrO, reflux, 36 hours,
66
857o.
Scheme 30.
Hence the preparation of a-alkynyl-o>functionalised linker-spacer compounds was
accomplished using standard chemistry in good yields. It was envisaged that alkynyl alcohol
59 would be used in PdCC reactions (with the hydroxy function subsequently being convefted
to an alþne), alkynyl acid chloride 62 and alkynyl iodide 67 in coupling to labels which
inco¡porate a nucleophilic group, and alkynyl amine 66 for coupling to electrophilic groups
of labels. The use of these compounds in the preparation of label-spacer molecules is
described in the remaining sections of this Chapter.
Results and
Discussíons
33
Chapter 2.2. Synthesis of Fluorescent Label-spacer Molecules.
The coupling step between label and spacer molecules for the construction of
fluorescent label-spacer adducts may occur via a palladium catalysed coupling of a terminal
aþne of the spacer with a halogenated derivative of the pyrene 7 moiety (Scheme,18,
Introductíon), or via nucleophilic/elecrophilic interactions for the acridone 12, fluorescein 5
and dansyl 10 moieties (Schemes 19 and
20,Intoduction). Previous works
had est¿blished
conditions for the preparation of the acridone label69 and pyrene label78, however the
yields were only moderate. Hence an improvement in yield for the preparation of 69 and78,
and the development
of conditions suitable for the preparation of label-spacer adducts based
on the fluorescein 5 and dansyl derivative 10 molecules were the objectives of this section.
The acridone label 69, as it has previously been synthesised easily in 59Vo yield
(Scheme 31, Conditions A), was selected to be a test label for optimising conditions for the
tabelling reactions with the halogenated and triflated biomolecule derivatives. The reaction
was repeated with 1-iodoundec-10-yne 62 as the alkylating agent in an attempt to improve the
yield of 69 (Scheme 31, Conditions
B).
TLC of the reaction mixture between 12
and NaH
in
DMF after stirring for 60 minutes at 50' suggested formation of the anion of 12 as shown by
the disappearance of the spot coffesponding to 12 at
appearance
&
0.17 (EtoAc/hexanes 40160) and the
of a new spot on the baseline. Also, the reaction mixture turned a fluorescent
yellow-green which is consistent with formation of the anion6s. Addition of 62 dropwise over
10 minutes discharged the fluorescent colour and gave a dark green solution. TLC of the
reaction mixture after 60 minutes stirring at room temperature showed a new spot at & 0.69
corresponding to product 69, alwge spot corresponding to 12 and the absence of the baseline
spot. After workup and removal of traces of DMF under vacuum, 69 was recovered in237o
yield; the balance of 12 appeared to be reprotonated. This ¡esult suggested that 62 was
reacting via an E, process and reprotonating the anion of L2. N-Alkylacridone derivatives
have also been synthesised in phase transfer catalysed reactions6, the best yields and mildest
conditions reported by Nishi et. dl.6' TLC of the reaction mixture after dropwise addition of
62 to a stirred mixture of 12,507o aeueous KOH, benzylriethylammonium chloride (0.02 eq)
and 2-butanaone (Scheme 31, Conditions C) at 80' showed after 6 hours a large spot for 69
Results and Discussíons 34
and the absence
of 62. The product was recovered after an easy workup \n 59Vo yield
high purity; the remainder of the anion
of
and
in
12 presumably remained in the basic aqueous
layer. The spectral data were identical with those obtained previously, although the melting
point of 95-97" was higher
(1it.64
88-90'). Although the yield of 69 was not significantly
different to the previous work, these reaction conditions were preferred as isolation of the
ptue compound was easiest.
o
_______>
I
H
t2
Conditions
Conditions
Conditions
69
A: (i) NaI{, DMF, 50" (iÐ HC=C-(CH)rBt;59Vo.
B: (i) NaII, DMF, 50'. (iÐ Iodide 62;237o.
C: Iodide 62 (I.5
eq), BzEgNCl (0.02 eq),507o KOH, 2-butanone;59Vo
Scheme 31.
The pyrene label-spacer adduct 71 has been synthesised previouslye in 60Eo yietd by
the palladium catalysed coupling of l-bromopyrene (70) and undec-10-yn-1-ol (59) (Scheme
32, Conditions
A). The moderate yield is typical of the cross coupling
reaction between aryl
bromides and terminal alkynes under these conditions. Piperidine, when used as the reaction
solvent and base, has been shown to enhance the slow oxidative addition step of the aryl
bromide to the palladium (O) catalytic species and give an increase in yieldae. Reaction of 70
and 59 with palladium and copper catalysts in piperidine (Scheme 32, Conditions
B) gave,
after overnight reflux and workup, 71 in a yield of 72Vo. An alternative system for the
activation of aryl bromides using pynolidine has also been reportedso. Reaction of 70 and 59
with only
a
palladium catalyst in pyrrolidine at 80" for 20 hours (Scheme 32, Conditions C)
and workup afforded
7l in a good yield of 79Vo. The more reactive conditions
best use of the alkyne; less is needed
if
also enable
the coupling occurs quickly as the slower, competitive
Results and
Discussions
35
homocoupling process is not favoured. The spectral data were identical with those obtained
previously.
H
->
70
7t
Conditions A: 59 (2.5 eq), Pd(PPh3)2CL (0.05 eq), CuI (0.1 eq), PP\ (0.1 eq), EgN,
pyridine, 90", 24 hours; 607o.
Conditions B: 59 (1.2 eq), Pd(PPh3)4 (0.025 eq), CuI (0.05 eq), PPh3 (0.05 eq), piperidine,
reflux ON;727o
Conditons C: 59 (1.2 eq), Pd(PPh3)4 (0.025 eq), pyrrolidine;797o.
Scheme 32.
The unsaturated pyrene label 74 was synthesised in good overall yield (617o) from 71.
Oxidation with PCC (Scheme 33a) gave the aldehydeT2 in excellent yield. Synthesis was
tH l.Ilt4R spectra of a resonance at õ
confirmed by IR (v^,* 1720 cm-l) and the appearance in
9.75(t,IIH,J 1.8H2)indicativeof thealdehydicproton. Conversion olT2totheterminal
alkyne 74 was via the method of Corey and Fuchs6t. Treatment of CBro with PPh, in
at
-15'for 30 minutes (Scheme 33b)
CIIC!
gave rise to an orange solution which is thought to
contain the ylid BrrC=PPhr. Addition of 72 to the reaction mixture and stirring for 60
minutes at 0'allowed attack on the carbonyl group via a Wittig-type process to give 73. TLC
(10/90 EtOAc/hexanes) of the dark brown reaction mixture showed the absence of 72 at about
&
0.20 and a new fluorescent spot at about
&
0.80 corresponding to 73.
Dehydrohalogenation and halogen exchange of 73 facilitated by n-Buli in TFIF (30 minutes
at -78' and room temperature for 2 hours), followed by protonation of the resultant lithium
acetylide with saturated NH.CI solution (Scheme 33c) gave the alkyne 74 in good yield.
Synthesis was confirmed by the disappearance in the
tH NMR of the dibromoalkene proton
resonance; a strong sharp absorption in the IR at 3300 cm-l and a new resonance at ô 1.95 (r,
IH, J 1.8 Hz) were both indicative of
a terminal alkyne.
Results and
Discussions 36
(CH2)s:
-R
arbc
+
71: R = CH2OH
72: R = CHO
73: R = HC=CBrz
74
a. PCC (1.5 eq), NaOAc (2eq),CIl2Clr;96Vo.
b. CBro, PPh3, CftCL, -10".
c. (i) n-Buli, TTIF, -78'to RT. (iÐ NH4CI (sat),647o (overall for steps b and c)
Scheme 33.
cHtlo-R
a
----------->
(CHz)ro-
brcrd
->
7l
75: R = CFI2OH
76: R = CHO
77: R = HC=CBrz
a. H", 57o PdlC, F;tO Ac, 89Vo.
b. PCC (1.5 eq), NaOAc (2 eq), CrtCL;89Vo.
c. CBro, PPh3, CFLCL,-1O";80Vo. d. (i) n-Buli, TFIF, -78'to
78
RT. (iÐ NH4CI (sat),717o.
Scheme 34.
Preparation of the saturated analogue 78 required reduction of the internal alkyne of
71 to give the saturated analogue 75. This was readily achieved by stirring
7I with a 5Vo
Pd/C catalyst under a hydrogen atmosphere (Scheme 34a). Filtration of the catalyst, removal
of the solvent and recrystallisation gave the saturated compound in excellent yield.
Conversion of 75 to the alkyne 78 via the aldehyde 76 was by the method for the unsaturated
Results and
Discussíons
37
analogue 74, with the yields being comparable. The spectral data for the alkyne were
identical to those obtained previouslye.
Attachment of a spacer molecule to fluorescein 5 to give a label-spacer adduct could
theoretically be achieved via either the carboxylic acid
ûo
give an ester or amide, or alkylation
of the phenolic hydroxyl to give an ether. However the small pKa difference between the
acidic and phenolic protons (pK, = 5.05,
pÇ = 7.00)ue, and lactone
isomerisation to 79 in
non-polar solvents or under acidic conditions makes selective alkylation of either group
difficult to
achieveT0. Also, the unreactivity of the carboxylic acid to carbodiimide réagents
such as DCCI prevents attachment of the spacer via an amide linkage.
A possible approach
was then to prepare the known fluorescein methyl ester 80, which should be easily separable
from other products in the reaction mixture, and to subsequently alkylate the phenolic
hydroxyl with the iodide 62.
H+
_.------------.
-
o
o
80: R=H.
IüSO. (cat.), MoOH, reflux T2hours;257o.
Scheme 35.
Results and
Discussions
38
The methyl ester of fluorescein 80 was prepared by refluxing 5 in MeOH with IISO4
as
catalyst lor T2hours (Scheme 35). The mono- and dimethylated byproducts which formed
were removed by dissolving the crude product in
lM NaOH solution, extracting
the aqueous
solution with EtOAc to remove the less polar dimethylated compounds, reprotonation with
l07oHClto precipitate the crude product,
and
finally recrystallisation from MeOH. The
methyl ester's identity was confirmed by mass spectrometry (M. 346) and melting point
(282";lit.72 282"), and was recovered in25Vo yield.
Alkylation to give the label81 was first attempted by deprotonation of 80, and
reaction of the anion with the iodide 62 (Scheme 36). Addition of NaH to a suspension of 80
in DMF caused effervescence and the formation of
a dark red
evolution of gas had ceased and the substrate was in
a dark
mixture. After
15 minutes the
red solution; presumably the
phenolic anion had formed. Compound 62 was added dropwise and the mixture stired for 60
minutes, then TLC (EtOAc) showed large spots for starting material at Rr 0.10 and product at
& 0.24. After workup and recrystallisation
the product was recovered in 367o
yield. A
molecular ion at mlz 496, a strong sharp absorption in the IR at3296 cm-t 1H-C= stretch) and
a
tH NMR resonance at 4.06 (t,2IF.', J 7.0}J2, CHr-OAr) confirmed alkylation had occured.
o
o
CO2Me
CO2Me
-->
80
Conditions
Conditions
A:
B:
81
(i) NaH (1.1 eq), DMF. (ä> 62 (1.1 eq).
62 (1.5 eq), K,CO, (2 eq),2-butanone, reflux 24 hours.
Scheme 36.
As presumably the starting material evident by TLC in the previous reaction was
reformed by the abstraction of a proton from 62,
a base
mild enough to exist with 62 and yet
deprotonate 80 was required. With an increase in the amount of 62, a higher yield should
Results and Discussioru 39
result. Reaction of 80 with 62 andÇCO, in 2-butanone at reflux overnight (Scheme 36'
Conditions B), workup and recrystallisation gave compound 80 in a good yield of 79Vo. The
product prepared by this method has the disadvantage that the most fluorescent form of
fluorescein, in which the phenolic group is deprotonated, is not available. Even so,.the
neutral form is sufficiently fluorescent such that a modified phosphoramidite 82 which
included an O-alkylated fluorescein ether methyl ester has been used to label
oligodeoxynucleotidesT3 successfully.
MeO
o
o
-o
82
Attachment of a spacer molecule to a functionalised fluorescein derivative, such as
S-aminofluorescein 83 allows the highly fluorescent phenolic anion to be formed. Hence
labe|84 was synthesised by the reaction of 83 with acid chloride 67 in pyridine (Scheme37).
After dissolution of 83 to give
a deep green
at room temperatue for 48 hours,
0.33, a small byproduct spot at
&
solution, 67 was added dropwise. After stirring
TLC (10/90
MeOIlCflC!)
showed a trace of 83 at
\
0.87 and a large spot corresponding to product 84 at Rr
0.54. After workup the product was recovered in 657o yield
as bright orange crystals.
lH NMR spectroscopy in
Acylation was confirmed by mass spectrometry (M.511) and
I2H16-DMSO, which showed fesonances atõ2.37 (t,2H.,J 7.3, CH2-CONH) and ô 2.73 (t,
1H, ,f 2.5 Hlz,
H-G).
Also
8¿t
was shown to exist in the lactone form 85 in DMSO. The
lH spectrum for the non-symmetrical, open chain form 84 would be
aromatic part of the
expected to have 12 resonances, each of which integrates to
lH (for the aromatic and acidic
Results and
protons). However only
8 resonances (4
Discussions 40
with integraúon 2H, the remainder lH) are
observed, which is consistent with the plane of symmetry in 85. In addition, the
13C
NMR
spectrum showed 25 resonances tather than 31, which is again consistent with 85. This facile
isomerisation explains the fact that a solution of 84 in a protic solvent (eg water, MeOH,
EIOH) is coloured and fluorescent whereas a solution of 84 in an aprotic solvent (eg EtOAc,
DMSO) is neither.
H
H
_____>
-l
84
83
85
Acid chloride 67 (1.2 eq), pyridine, 48 hours, room tempelature; 657o.
Scheme 37.
The dansyl label 87 was prepared by reaction of dansyl chloride 86, the aminoalkyne
66 and EqN in CÍI"Clrat room temperature (Scheme 38). After stirring for 60 minutes
(although presumably the reaction was over much sooner) TLC (20180 EtOAc/hexanes)
showed the absence of non-fluorescent 86 at
\
0.71 and a new fluorescent spot at
&
0.42
corresponding to product. After chromatography 87 was recovered in 727o yield with MS
lH NMR showing a resonance at õ 4.59 (bt, LH)
showing a molecular ion at mlz400, and
consistent with the proton of the sulphonamide group.
With the preparation of the dansyl label-spacer adduct 87 all of the selected
fluorescent moieties had been attached to spacer molecules in average to good yields.
Fluorescence data of the compounds is shown in Table 2, and coupling reactions with the
biomolecule derivatives are described in Chapter 3.
Results and Discussions
--------J>
-o
86
87
(1
(1
eq), CIlCl2i727o
eq), EqN
Amine 66
Scheme 38.
Table 2. Fluorescence Data for Label-spacer Adducts.
n-
À-ur nm
Compound
Solvent
69
EtOH
385
42L,444
74
cHC13
364
386,396,406
78
cHC13
343
377,397,416
81
EtOH
489
5t7
84
EtOH
482
516
87
EtOH
337
506
À"*
4l
Results and
Discussions 42
Chapter 2.3. Synthesis of Time Resolved Fluorescence
Label-spacer Molecules.
The synthesis of a time resolved fluorescence (TRF) reporter compound 92 required
the incorporation of an alkynyl-alþl chain-modifred-1,10-phenanthroline ligand 91.into
rurhenium (II) brs-l,lO-phenanthroline dichloride 90. Retrosynthetic analysis (Scheme 39)
showed that palladium catalysed coupling of the alkynol59 with S-bromo-1,10-phenanthro-
line 88, and subsequent functional group interconversion from a terminal alcohol to a
terminal alkyne was necessary. The internal alkyne of the adduct 91 can either be reduced to
give a saturated link to the 1,lO-phenanthroline moiety, or retained to modify the fluorescent
behaviour of the label.
Br
HteoH
88
+
89
:-(CH2)eoH
59
0:
l0:
9I
+
R(ptrcn)2C12
90
92
Scheme 39.
The preparation of 5-bromo-1,10-phenanthroline 88 has been achieved via a Skraup
synthesis from 8-amino-6-bromoquinoline?a andby the bromination of l,lO-phenanathroline
Results and
Díscussions 43
40 in fuming sulphuric acid (607o oleum¡7s lScheme 40), both methods reporting an average
yield. The latter method was preferred
as the substrate and reagents were readily available,
and did not require the toxic arsenic pentoxide.
Initial attempts under the reported conditions
gave only a poor yield, with a large amount of dark red byproduct (which was not
characterised). Heating anhydrous 40 at 120" at0.01 mmHg for three hours prior to reaction
under the previous conditions to ensure anhydrous starting material reduced the amount of
byproduct, and increased the yield to 52Vo. The product was purified by chromatography on
alumina as pronounced str,eaking occurred on silica TLC; slow elution from a short column
gave the best separation from unreacted starting material. Mass spectrometry confrrmed
rH NMR were in accordance with the
monobromination (m/22581260); melting point and
literatureT5.
NN
€
NN
40
88
Br, fuming IüSO.,
125", 12 hours; 52Vo.
Scheme 40.
Previous palladium catalysed coupling reactions of bromo-substituted
1,lQ-phenanthrolines have used CuI as a co-catalyst76, however complexation of Cu(I) to the
phenanthroline moiety reduced the yield of the reaction. The use of sonication to disrupt
complexation, and treatment with aqueous KCN were necessary to allow a good yield. The
use of copper iodide as a co-catalyst is not necessary (introductíon, p 20)
if the substate
can
withstand the higher temperatures of pynolidine at reflux, hence 88 was reacted with alkyne
alcohol 59 to give the adduct 89 in 66Vo yield (Scheme 41). Coupling was conf,rmed by MS
(M. 346), the absence of the terminal alkyne triplet at ô 1.97
at õ 2.68 (2H,J
7.lHz) indicative of Ar-:-CÉ12.
and the presence of a new triplet
Results and
Discussions 44
cHreoH
--------_>
NN
NN
88
89
59 (l.zeq), Pd(PPq)4 (0.05 eq), pynolidine, 80', 6 hours; 667o.
Scheme 41.
Treatment of 89 with
5Vo
Pd/C under an atmosphere of hydrogen overnight (Scheme
42, Conditions A) showed no reaction. Increasing the amount of palladium to lÙVo and
srirring for 48 hours (Scheme 42, Conditions B) gave a mixture of starting 89 and the alkene
94 (in a ratio of approximately 1:7), as indicated by vinylic resonances at ô 6.02 (dt, J
7.5¡¿2, C¡I2-HC=) and ô 6.77 (dd,
J
lL5,
11.5, L.ZHz, AI-HC=). As the vinylic coupling constant
was 11.5 lH2,94 was assumed to be the expected Z isome/7. Increasing the reaction
temperature to 55' and stirring ovemight removed the alkene resonances, however the
aromatic region was now very complex, indicating a mixture of products. Presumably
complexation of the l,lO-phenanthroline moiety to the catalyst was the cause of the sluggish
reaction. Increasing the temperature to decrease complexation caused overreduction.
Protonation of amines is a common way of preventing complexation to the catalyst in
hydrogenation reactionstt, hence reaction of 89 under acidic conditions (Scheme 42,
Conditions C) gave the saturated compound 93 in787o yield. Mass spectrometry showed a
molecular ion at m/2350, and
lH NMR showed the absence of the resonance at õ 2.68, and
a
new resonance at E 299 (t,2H', J 7 .7 Hz, CHr-At) which were consistent with hydrogenation
having occurred. The remainder of the starting material appeared to be converted to a red
polar compound which could not be eluted from the top of the alumina column used for
purification. Repeating the reaction using sulphuric acid gave
a
yield of 527o, and with
chloroacetic acid a yield of 527o was obtained. The reduction step occasionally did not go to
Results and
Discussions
45
completion (even with catalyst renewal) and in such cases the partially reduced compound
could not be separated by column chromatography.
H
HO(CHre
r-oH
+
--------_:>
NN
N
N
N
94
93
89
Conditions A: [t, SVoPdlC, MeOH, OÆ'{.
Conditions B: FIr, lÙ%oPdlC, MeOH,48 hours.
Conditions C: Hr, 57o PdlC, lÙVo IJCI, l!'feOIJ; 7 ïVo.
Scheme 42.
Conversion of the alcohol 93 to the aldehyde 95 using PCC (Scheme 43, Conditions
A) was unsuccessful. TLC analysis of the reaction mixture showed only
a
large amount of
baseline material, suggesting that complexation of the 1,lO-phenanthroline moiety to the
reagent had occurred. Recently, TEMPO (2,2,6,6-tetamethyl-l-piperidinyloxy free radical)
has been used as a catalyst for the large scale oxidation
of alcohols to carbonyl
compoundsTe.
The advantages of this method have been reported to include no moisture sensitivity, ease of
workup and ease of reaction on
a
large scale. When 93 was reacted with NaOCl, NaBr and
TEMPO (Scheme 43, Conditions B)
a
low yield of 95 was obtained, with TLC analysis
showing a small amount of starting material remaining. Oxidation was confirmed by the
absence in the IR
of
broad O-H stretch between 3600 and 3100 cm-r, and a new intense IR
absorption at1724 cm-1 consistent with a C=O stretch. The majority of the starting material
was presumably converted to the corresponding acid, which was not isolated from the
biphasic reaction mixture. Reverting to the standard Swern oxidationso using DMSO, oxalyl
lH
chloride and EqN (Scheme 43, Conditions C) afforded the aldehyde 95 in good yield;
NMR of the crude product showed conversion was effected relatively cleanly. The aldehyde
Results and
Discussions 46
was pattially unstable to chromatography on alumina hence the isolated yield was average,
and so the crude aldehyde was used in the next synthetic step.
(cH2)1r-oH
NN
93
Conditions
Conditions
Conditions
o-cHo
--->
N
95
A: PCC (1.5 eq), NaOAc (2 eq), CIJ"CIr,0Vo.
B: TEMPO (0.1 eq), NaOCI (1.1 eq), NaBr (1.1 eq), CIl"O,HrO;387o.
C: CICOCOCI (1.1 eq), DMSO (2.2eq),EgN (5 eq), CI!C!,-78" toRl;717o
Scheme 43.
Conversion of 95 to the alkyne 91 was to occur via the method of Corey and Fuchs8l,
which involves reaction of 95 with the Wittig-type roagent derived from CBro and PPhr to
give the dibromoolefin 96, followed by dehydrohalogenation and halogen exchange with 2
equivalents of
n-Buli to give the lithium acetylide and protonation to give the alkyne
(Scheme 44). The dibromoolefin 96 was recovered in a poor yield of 35Vo, atd contaminated
with triphenylphosphine oxide (in a ratio of 95:5), which could not be removed by repeated
tH resonances at ô
chromatography or recrystallisation. Formation of 96 was confirmed by
6.34
(t,lH, HC=CBrr)
and ô 2.04 (q,2H, CÉ1r-CH=CBrr).
-CHO
(CH2hsCH=CBr2
(Ð
(Ð
_------>
NN
95
NN
96
-----i>
9l
(i) CBro, PPh3,-10o,3 hrs, 35Vo. (ä) (a) n-Buli (2.2eq), TTIF, -78o- RT,2 hrs; (b) NH.CI (sat)
Scheme 44.
Results and
Discussions
lH NMR analysis of the reaction mixture of 96 with n-Buli (2.2eq) showed
47
a
different and complex aromatic region, suggesting the lithium reagent had reacæd with the
1,lg-phenanthroline moiety to give many products. Nucleophilic addition of alkyl lithium
species to C2 and C4 of pyridine systems is well knownt2, and occurs readily at roor-n
temperature. Repeating the reaction but quenching at -78'again gave
a,
complex aromatic
tH l.IIvfR spectrum and many products by TLC. Given the poor yield and
region in the
purification problems of the previous step an alternative method for the conversion of 95 to
9l
was necessary.
(Ð, (Ð
-CHz-Br +
o
H2N-CH2- [--oet
OEr
+
(trÐ
98
97
(i) P(OEÐ3, p-xylene,
A,,
48Vo. (iÐ NrHo.HrO, AcOH. (iii) NaNO r, AIOH, 63VoScheme 45.
Dialkylphosphinodiazomethanes such as 98 have been shown to convert aldehydes to
alkynes in average to good yields83. Compound 98 was readily prepared by the reaction of
commercially availableN-(bromomethyl)phthalimide 97 with triethylphosphite, removal of
the phthalimido group with hydrazine hydrate and diazotisation with NaNO2 under acidic
conditions (Scheme
45)*. Although adiazo compound,
98 is relatively stable due to the
elecgon withdrawing influence of the phosphino group, and purification by distillation was
possible. The postulated mechanism for the reaction (Scheme 46)85 of 98 with aldehydes
involves nucleophilic addition of the diazo compound anion 99 to the carbonyl gtoup, and
formation of a Wittig-type intermediate 100 which loses potassium diethyl phosphate to give
a diazoalkene
101. Loss of nitrogen from
l0l
results in a alkylidenecarbene L02, which
undergoes hydrogen migration to give the alkyne 103.
Results and
K+
o
il
R-CH
R
_-------->
-----------:>
N
N2
99
Díscussions
R
C:C: Nz +
H 101
100
48
1
(EIO)2PO-+K
-N2
R-C:C-H
R
<-
H
C:Cl
L02
103
Scheme 46.
Hence treatment of 95 with 98 was attempted (Scheme 47, Conditions A), and gave
the alkyne
glin 477o yield. Synthesis was confirmed
by
tH NMR resonances at 1.95 (r, lH,
HC=C) and2.23 (dt,2IH,CH2-G{H), and IR absorptions at 3300 cm-l (s, H-C: str) and
2060 cm-l (w,
GC srr). Recently the use of dimethyl-(l-diazo-2-oxopropyl)phosphonate
(CH3C(O)C(Nr)P(OXOMe),) for the conversion of aldehydes to alkynes under mild
conditions has been describeds6
. Using this reagent
for the conversion of 95 to 91 (Scheme
47, Conditions B) gave a yield of 607o.
(cHùocHo
NN
Conditions
A:
Conditions
B:
N
9s
9L
(Ð 93 (1.1 eq), KOBu'(1.1 eq), TTIF, -78',10 mins;
(iÐ RCHO, 18 hrs,T to RT.
CIIC(O)C(Nr)P(OXOMe), (1.1 eq), KrCQ (1.1 eq), MeOH, 0",12 hours
Scheme 47.
Results and Discussíons 49
Using an analogous route the unsaturated ligand 105 was synthesised by the
procedures developed for 91 (Scheme 48). Alcohol Sg was converted to the aldehyde 104
using a Swern oxidation, and subsequent reaction with 98 gave the alkyne 105. The IR
spectrum of 104 showed the absence of the absorption between 3600 and 3100 cm-l-(O-H
stretch), a strong absorption atl722cm-t
spectrum atõ 9.72 (t, J
l.6Hz).
1C{)
and the aldehyde resonance in the
tH l'[lr'IR
Conversion to the alkyne 105 was confirmed by the
characteristic absorption in the IR at 3300 cm-t çH-G sE), absence of C=O absorption at
1722 cm't and the alkyne proton resonance at ô 1.95 (t, J 2.6IJ2).
H
(Ð
(Ð
______>
-_------->
NN
N
N
N
104
89
10s
(Ð (COCI), (1.1 ee), DMSO (2.2eq),EqN (5 eq), -78" to RT,697o.
(iÐ (a) HC(\)P(OXOEÐ, (1.1 eq), KOBu'(1.1 eq), TTIF, -78', 10 mins;
(b) RCHO, stfu 18 hours, then warm to RT, 607o.
Scheme 48.
Formation of the ruthenium complex 92 was achieved by stirring 91 with
Ruþhen)rCl 90 in a mixture of MeOH
and
tlO
at 40"
for 48 hours (Scheme
49).
The dark
purple colour of the neutral ruthenium (tr) complex in solution changed to a deep red-brown
as the reaction progressed.
After filtering out
a
black precipitate (presumably ruthenium
metal), concentration of the reaction mixture and addition of aqueous NHoPFu, red-orange
crysrals of the rurhenium (II) salt 92 were obtained. Both
lH NMR and TLC showed
impurities present however attempted recrystallisations from various solvents were
unsuccessful: the compound continued to oil out of solution. Purification was effected by
chromatoglaphy on alumina (silica gel caused decomposition) and the product was recovered
in an average yield o1687o. LSIMS showed an intense peak at mlz95l1r02RuM2*.PFr-¡,
Results and
confirming incorporation of
9l into the ruthenium complex.
Discussiors 50
As the parent rris-1,10-
phenanthroline ruthenium (II) complex is chiral, formation of diastereomers upon addition of
the non-symmetrical modified ligand was expected. The
tH NMR showed two overlapping
triplets atõ 3.22 and õ 3.24 (total integration two protons) instead of a single triplet.for the
methylene adjacent to the aromatic moiety, confirming non-separable (by column
chromatography) diastereomers. As the coupling properties of the alkyne under palladium
catalysis were of interest, separation by a higher resolution method (i.e. HPLC) was not
attempted.
l0
Ru(phen)2Cþ
90
r/N
€
+
N
91
zPF6
10
92
MoOIVIIO (I:2), 40o, 48 hours,
687o
Scheme 49.
With the incorporation of the modified 1,lO-phenanthroline ligand 91 the synthesis of
the target reporter compound 92 was complete. Fluorescence spectroscopy of a solution of 92
showed a maximum at578 nm (),.* = 449
nm).
The unsaturated ligand 105 was not
incorporated into a ruthenium complex due to time constraints. Reaction of the reporter 92
with a phenylalanine derivative under palladium catalysis is described in Chapter 3.1
(Coupling of Labet-spacer Molecules to Aminoacid Derívatives).
Results and
Discussions
51
Chapter 2.4. Synthesis of Biotin'spacer Label.
A common method of synthesising biotin conjugates is via reaction of an activated
biotin ester (e.g. the N-hydroxysuccinimide ester 106) with an amine to give aN-substituted
biotin amide derivative. Preparation of 106 by reaction of 41with NHS and DCC in DMF
(Scheme 50) gave the product in average yield87. The melting point and spectral data of 106
were consistent \t/ith the literaturets.
H
\
H
N
_____> o
S
H
H
\
H
H
4t
Scheme 50. NHS, DCC, DlvÍF;5lVo
106
Displacement of the NHS group by the amine 66 occurred readily in DMF (Scheme
51). Filration to remove precipitated NHS, removal of the solvent and purification of the
residue by flash chromatography and recrystallisation from MeOtVfLO gave the biotin
undecynyl amide 107 in very good yield. Synthesis was confîrmed by MS (M. 393); the
NMR data were consistent with the expected structure. Unexpectedly, when testing for
a
suitable recrystallisation solvent, the compound was found to gelate EtOAc and other low
polarity organic solvents. An investigation into the gelling properties of 107 and other biotin
analogues is reported
in Chapter 4.
o
o
€
H
\
o
N
H
(9tt)o
H
I
H
106
Amine 66 (1 eq), DMF, RT, 15 hours; 907o.
Scheme 51.
107
(CH2)e:
Results and
Discussions 52
Chapter 3. Preparation and Coupling of
Biomolecules
Chapter 3.L. Coupling of Label-spacer Adducts to Aminoacid
Derivatives
This section describes the preparation of the halogenated and triflated amino acids
108, 109, 110 and 111, and their behaviour under PdCC conditions to a selection of the
developed label-spacer adducts. Also, the reaction of propargyl glycine derivative 126 with
l-bromopyrene and l-iodopyrene is reported. Protection of the amino and carboxyl
functionalities of the amino acid derivatives, although not necessary during palladium
catalysed couplings, was undertaken for reasons of ease of purification and characterisation.
Esterification of the amino acid derivatives with MeorVSOCl, gave the methyl ester
hydrochlorides (Scheme 52(i)), generally in excellent crude yields. Removal of the solvent,
treatment of the crude products with benzoyl chloride under Schotten-Baumann conditions
(Scheme 52(Íi)) and column chromatography on silica gel followed by recrystallisation gave
the protected amino acids in avemge to good yields.
+
"K"
H¡N
COz-
oil
PhCN
-->
H
CO2Me
(i) MeOH, SOCI2. (iÐ PhCOCI, K2CO3, CII2C12,Í12O.
Product
yleldVo
4-iodophenylalanine (46)
108
73
3-iodotyrosine (47)
109
59
tyrosine (48)
110
55
S-hydroxyltryptophan (49)
111
50
Amino acid
Scheme 52.
Results and
Discussions 53
Hydroxy amino acids 110 and 111\ryere converted to the triflates
N-phenyltriflimide (PhNTf2) andEt N in
Cflcl
ll2
and 113 using
(Scheme 53). Purification by flash
chromatography and recrystallisation gave the products in high yields. Triflate 112 was a
known compoundte and melting point and spectral daø were in agreement. Formatþn of 112
was confirmed by MS (M.470) and IR (absence of O-H stretch).
H,o
oil
PhCN
CO2Me
Tf
H,o
I
H
CO2Me
lL2:
110
997o.
H
H
N
I
I
-------+
PhCN
I
H
OH
CO2Me
orf
CO2Me
ll3:
111
917o.
PhNTf2 (1.1 eq), EqN (1.1 eq), Ct!C12,0o to RT, overnight.
Scheme 53.
The reaction of the acid derivatives with the label-spacer adducts were first attempted
under standard conditions, which are shown in Scheme 54. Modifrcations to the conditions,
such as changing temperature, catalyst, solvent and/or base were made
if the coupled product
was not obtained in an acceptable yield. Reactions of the triflate 112 with the acridone label
69
are described
first.
Ar-X
+
Ar-X (X =I, OTÐ (1.0 eq),label (1.2 eq), Pd(PPq)4
Scheme
Ar---label
(0.1 eq), CUI (0.2 eq), EgN, DMF' RT
54: Standard Conditions
Results and
Reaction of
ll2
with 69 under standard conditions
Discussions 54
at room temperature (Scheme 55,
Conditions A) showed no coupled product (by TLC comparison of product synthesised from
the iodide 108) after 60 minutes. Increasing the temperature sequentially to 35', 50'and 60"
after 60 minute periods again showed no reaction. The only new fluorescent spot was due to
the homocoupled alkyne dimer l-14 in increasing quantity; triflate 112 was still present.
Repeated reactions
with increasing amounts of catalyst (lÙVo, l5%o and3D%o), and increasing
temperature again gave only the homocoupled product.
-(CHr)n------:-------
-(CH)s -N
tl4
As 112 was still present in the reaction mixture, this showed that homocoupling of the
alkyne was occurring at a faster rate than formation of the cross-coupled product. In the cross
coupling reaction, the rate determining step usually is either the oxidative addition of the
electrophilic carbon species to PdL, or transmetallation by the nucleophilic carbon onto the
pallad.ium (II) intermediate 115. The use of Pdrdbq/AsPh, catalyst system has been shown to
increase the rate of the transmetallation step as the rate of the ligand dissociation from the
intermediate 115 is increasedeO, hence facilitating formation of the transmetallated
intermediate
llt.
However TLC analysis of the reaction mixture between
ll2
and69
(stined at room temperature overnight) under these conditions (Scheme 55, Conditions B)
showed only alkyne dimer 1-l4
ll2
and 69 were still present. Increasing the temperature to
50" and stirring until the catalyst decomposed (about 30 hours in total) showed only an
increase in the amount of 114 and the absence of
ll2
and
69. A spot corresponding to the
desired coupled product 118 was not observed. Removal of the solvent and separation of the
residue by chromatography gave
ll4
in
617o
yield (from starting alkyne). Increasing the
Results and
Discussioru
55
amount of catalyst ¡o 707o with stirring at 50', and workup as previously gave only 114 in
67Vo y¡eld. The lack of coupled product 118 suggested that oxidative addition was probably
the rate determining step in this system-
L
I
oll
Pd
H,,,
L
I
PhCN
H
CO2Me
L
-orf
I
Pd
CO2Me
H
115
+
-L
Lt7
L
Iabel
CO2Me
I
H
116
The rate of oxidative additon of aryl bromides in palladium catalysed cross-coupling
reactions is relatively slower compared to aryl iodides, and so conditions reported to effect
efficient coupling of aryl bromides were
ll2
tested4e
lScheme 55, Conditions C). Reaction of
and69 at reflux under these conditions gave only unreacted starting alkyne (28Vo) and
the dimer 1-L4 (32Vo). The triflate was consumed in the reaction but the resultant
compound(s) were not identified. A large amount of baseline material was observed
suggesting the decomposition of
oil
ll2
under the reaction conditions.
H'o
PhCN
I
H
CO2Me
-_______)
PhCN
I
H
CO2Me
tt2
Conditions
Conditions
Conditions
Conditions
Conditions
Conditions
118
A: 69 (1.5 eq), Pd(PPh3)4 (0.1eq), CuI (0.2 eq), EqN, DMF.
B: 69 (2.5 eq),PÇdbq (0.025 eq), AsPh, (0.2 eq), CuI (0.1 eq), EqN, DMF.
C: 69 (1.5 eq), Pd(PPh3)4 (0.1 eq), CuI (0.2 eq), PPh, (0.2 eq), piperidine.
D: (i) Pd(PP\)4 (1.0 eq), DMF. (iÐ 69 (1.5 eq), CuI (0.2 eq), EqN; 687o.
E: 69 (1.5 eq), Pd(PPh3)4 (0.1 eq), Et N, DMF,90'.
F: 69 (1.5 eq), Pd(PPh3)4 (0.1eq), CuI (0.2 eq), EqN, DMSO; 867o.
Scheme 55.
Results and
Thus
it
appo¿trs that the
Díscussions 56
oxidative addition step of the aryl triflate is slow, which
allows the (normally) slower homocoupling process (which is catalysed by copper and
palladium speciese2) to predominate. This problem may be overcome by reacting the triflate
stoichiometrically with Pd(PPhr)o to form the o-bonded intermediate 115 (which is lhought to
be in equilibrium with the ionic palladium species 116e3), and is then reacted
with the label
under standard conditions.
Reaction of lL2 with a stoichiometric amount of Pd(PPhr)o in DMF (Scheme 55'
Conditions D) at room temperature gave rise to a green precipitate (which decomposed upon
exposure to the atmosphere). After 3.5 hours TLC analysis showed the absence of lLZ at Rr =
0.25 and a new spot at
&
= 0.05. As presumably the o-complex had been formed the label,
CuI and EgN were added. The green precipitate was immediately consumed and the reaction
mixture turned dark brown. After stirring at 50'for 30 minutes, TLC analysis
(EtOAc/hexanes 50/50) showed the absence of the low
unreacted 69
\
spot, and spots corresponding to
(& = 0.63), dimer 114 (& = 0.37) and coupled product
118
(& = 0.22). After
separation by flash chromatography 118 was recovered in 687o yield;487o of the label was
converted to the dimer 114. The structurc of the product was confirmed by mass
tH spectrum was a superimposition of the
spectrometry (M. 626); the appearance of the
spectra
of
ll2
and 69, apart from the absence of the resonances for the terminal
adjacent methylene, and a new resonance at ô 2.40
alþrte
and
(t,2H,J 7.0IJ2) consistent with a
methylene attached to an ethynylbenzene group.
Although the stoichiometric coupling of triflate 112 was successful in average yield,
conditions for catalytic couplings were still sought as palladium reagents are expensive, and
isolation of the product is easier when a catalytic amount is used. Chen and Yangea reported
that phenyl triflate was coupled with a variety of terminal alkynes under palladium catalysis
in DMF at 90"; no CuI was used (Scheme 55, Conditions E). Howeverreaction of 112 with
69 under these conditions resulted in alþne dimer 114 in 337o yield of starting alkyne and
small amounts of unreacted starting triflate and label. The remainder of the starting materials
appeared to undergo decomposition.
Results and
Díscussions
57
DMSO has been used as a solvent in coupling reactions where the reaction
temperatuÞ has been raised to facilitate reaction of an unreactive substratees (Scheme 55,
Conditions F), and so reaction of lLZ with 69 at 70' gave the coupled product in 86Vo yield.
A smatl amount of alkyne dimer lL4
(3Vo) was also recovered. Repeating the reactlon
in
DMF under standard conditions (Scheme 55, Conditions A) with the temperature at 70'from
the start of the reaction resulted in a yield of 807o of the desired coupled product 118,
showing that the temperature of reaction is critical to the coupling. Presumably as the
temperature rises the rate of oxidative addition of the aryl uiflate to the palladium catalyst
becomes significant and hence can react further in the catalytic cycle to give the coupled
product. This approach is, however, limited by the thermal stabilities of the substate,
coupling compound and catalyst.
H
H
I
N
I
N
CO2Me
H
OTf
->
PhCN
I
H
CO2Me
119
113
Scheme 56.
A: Acridone
B: Acridone
label (1.5 eq), Pd(PPq)4 (0.1 eq), CuI (0.2), EtrN, DMF.
label (1.5 eq), Pd(PP\)4 (0.1 eq), CuI (0.2 eq), PPh, (0.2 eq),
piperidine; 87o.
Conditions C: (i) Pd(PPh3)4 (1 eq), DMF, 50'. (iÐ acridone label (1.5 eq), CuI (0.2 eq), Et N.
Conditions
Conditions
As 112 has been successfully coupled to the acridone label69, the reaction of
tryptophan triflate derivative 113 with 69 was explored by reaction under standard conditions
(Scheme 56, Conditions A) at 50". After 60 minutes TLC analysis @tOAc/hexanes 40160)
showed alkyne at
& 0.57, triflate 113 at Rr 0.16,
and alkyne dimer
ll4
at& 0.30. After
stirring overnight the amount of alkyne dimer had increased; other stafüng materials were
Results and
Discussions 58
still present. The reaction mixture developed a large amount of black precipitate (presumably
palladium metal) after 24 hours indicating the catalyst had decomposed. Separation of the
reaction mixture by flash chromatography gave the alkyne dimer in25Vo yield, atd757o of
the starting triflate was recovered. The balance of starting materials appeared to have
decomposed, as suggested by the large amount of low Rf material left on the silica column.
As the majority of triflate 113 was recovered in the previous reaction, 113 was reacted
with 69 in pynolidine at reflux (Scheme 56, Conditions B) in an attempt to increase the rate
of oxidative addition of the triflate to the active catalytic palladium species. After 5 hours at
reflux, TLC (40160 EtOAc/hexanes) showed the absence of 113 at &0.18, unreacted 69 at \
0.60, dimer 1-l4 at Rr 0.33 and a new fluorescent spot at Rr 0.11. Separation by flash
chromatography gave the coupled product 119 in a poor yield of 87o andthe alkyne dimer in
397o yield(calculated from sta¡ting alkyne). Mass spectrometry
(M. 689) and lH NMR
confirmed the synthesis of 119. Again, the balance of starting materials appeared to have
decomposed. The reaction was repeated at room temperature in an attempt to lessen
decomposition, however after overnight stirring 113 was completely consumed, and the only
fluorescent spots were due to 69 and 114. Evidently 113 was not stable under the reaction
conditions.
Given the success of the stoichiometric couplin g of 112,113 was stirred with an
equivalent of Pd@Phr)o in DMF (Scheme 56, Conditions C) at room temperaturc. TLC after
90 minutes showed no reaction, so the temperature was raised to 50". A green precipitate
slowly formed as the initial dark brown colour was discharged. After 4 hours, TLC showed
the absence of 113 at Rr 0.81 and a large spot at
\
0.14; a small amount of baseline material
was also observed. As the o-bonded intermediate had presumably formed, 69, CuI andEgN
were added, the green compound was consumed and the reaction mixture stirred for 60
minutes. TLC analysis of the reaction mixture showed the absence of the intermediate, a
large amount of 114, and no trace of product.
As reaction of
f-1.2
with the label occurred under standard conditions at higher
temperature in DMF and DMSO, 113 was reacted with 69 in DMF at70" (Scheme 56,
Conditions
A). TLC after 90 minutes showed starting
materials, and a small spot possibly for
Results and
Discussíons
59
product. After overnight stirring TLC showed the absence of 113, a large spot for the dimer
114 and a small fluorescent spot at low
Rf. The desired product 119 was recovered in a poor
yield of 2l7o after workup and chromatography (two passages through a silica column were
required due to the large amount of decomposition). The label dimer 114 was not is.olated.
It appeared unlikely that the desired product could be obtained in good yield without
modification of 113 (possibly protection of the indole nitrogen) or the label (functionalisation
of the alkyne to a morc reactive group, such as a borane or stannane derivative). These were
not considered, as it was desired to develop general reaction conditions which facilitated
labelling of all selected biomolecules and labels. Hence attempted coupling reactions of 113
were discontinued, and reaction of the 4-iodophenylalanine derivative 108 with labels
commenced.
Reaction of 108 with the acridone label69 proceeded at room temperature under
standard conditions (Scheme
57). The reaction was left overnight for convenience; TLC
@tOAc/hexanes 50/50) the next morning showed the absence of 108 at
fluorescent spots at
&
0.51 and 0.37 and a small fluorescent spot at
unreacted 69. Separation by flash chromatogaphy and
compound with
\
&
&
0.65, major
0.77 corresponding to
lH NMR analysis showed the
0.51. was the alkyne dimer 114; the compound
with
\
0.37 was the
product 118. Although the sizes of the spots on the chromatogram were similar, 118 was
recovered in an excellent yield of 96Vo whereas 114 was recovered in only 3Vo (ftom
conversion of starting alkyne).
I
PhCN
I
H
CO2Me
____-------:>
PhCN
H
CO2Me
108
118
69 (1.5 eq), Pd(PPq)4 (0.1 eq), CuI (0.2), Et N,DlvlF:967o.
Scheme 57.
Results and
Discussions 60
o
il
I
H
c(cH2)4
H
.H
H-
L20
3
H,,
PhCNI
H
CO2Me
NMø
l2l
OH
H,o
CO2Me
122
oil
H'"
Phc\r
H
I
CO2Me
lo
2PF6
123
Reaction of 108 with the biotin 107, dansyl87, aminofluorescein 84 and
ris-1,l0-phen- anthrolineruthenium
Ss labels all proceeded under standard conditions at
room tempetature in less than four hours to give the coupled products 120 (in 84Vo),
LZl
(82Vo),122 (757o) andl23 (65Vo yield) respectively. Analytical TLC showed the absence of
108 and new spots for the coupled products; the alkyne dimer was not observed in all cases.
Results and
Discussions 6l
purification by flash chromatography gave the labelled products in good to excellent yields,
and mass spectrometry and spectral data were consistent with the expected structures. As 108
proved to be a suitable substrate for coupling a wide range of labels with different
functionalities, testing of the iodotyrosine derivative 109 was commenced.
Reaction of 109 with the fluorescein tabel Sl proceeded at room temperature under
standard conditions (Scheme 58). The reaction was completed within 4 houn, as indicated
bytheabsenceofstartingmaterialbyTLC(Rf0.71,10/90MeoIVCrlC|)andanewmajor
fluorescent (1, = 365 nm) spot at &0.33. A small fluorescent spot presumably corredponding
to the alkyne dimer was also observed at Rr 0.21 but not isolated. Purification by flash
chromatography gave the labelted compound l24in9l%o yield. The identity of the product
was confirmed by MS (M.793) and the spectral data were consistent with the expected
rH spectra of a triplet at ô 2.48 (2H, J 7.0Hz)
structures, in particular the appearance in the
indicative of
oI
a
methylene group next to an alkyne attached to a benzene ring-
H,
PhCN
I
H
_------_>
CO2Me
oil
OH
H,n
PItCN
I
H
CO2Me
124
109
31 (1.2 eq), Pd(PPh3)4 (0.1 eq), CuI (0.2 eq), Et N, DlvlF:9lVoScheme 58.
Reaction of 109 with the biotin label 107 under standard conditions (Scheme 59)
again proceeded at room temperature. The reaction was complete within 2 hours, TLC
(10/90
MeOIVCIIC!) showing the absence of 109 at Rr 0.84, a major new spot at Rr 0.38
corresponding to product and a minor spot at
&
0.15 presumably corresponding to the alkyne
dimer. Purification by flash chromatography gave the labelled compound 125 in 76Vo yield.
Coupling was conf,rrmed by mass spectrometry (M. 690), and the spectral data were
consistent with the expected structure.
Results and Discussions 62
o
il
(cHreryc(cIJùq
H:
H
H
H-
_______>
H
CO2Me
I
H
109
L07
L25
(I.2eq),
Pd(PPh3)4 (0.1 eq), CuI (0.2 eq),
Et N,DlvlF:767o.
Scheme 59.
Wittr the successful labelling of 109, all of the aromatic amino acids selected had been
tested for labelling suitability under PdCC. With the exception of the uryptophan derivative
113 conditions were found where the substrates reacted readily with the labels, to give the
adducts in good to excellent yields and under mild conditions. The remaining amino acid
derivative to label was the propargyl glycine derivative L26, and these reactions are described
next.
Reaction of L26 with L-bromopyrene 70 at 55' overnight under standard conditions
(Scheme 60, Conditions A) gave the labelted compound 127 in 497o yield. TLC (60/40
EtOAclhexanes) showed the absence of 126 at Rr 0.36, unreacted 70 at
\
0.89, a new
fluorescent spot at Rr 0.29 (corresponding to product) and a large non-UV active spot at &
0.08 (presumably the alkyne dimer 128). Mass spectrometry (M* 383) confirmed the
coupling and NMR spectra were consistent with the structure. As the yield was average, the
reacrion was repeated in refluxing piperidine (Scheme 60, Conditions
B). TLC of the
reaction mixture after 60 minutes at reflux showed the absence of 70 and a fluorescent spot
for
l2l.
After chromatography 127 was recovered in
5LVo
yield. As the yield was not
significantly better, coupling of 126 to l-iodopyrene was attempted under standard conditions
(Scheme 60, Conditions
A). The coupled
product was obtained in a good yield of 72Vo after
stirring at room temperature overnight. Repeating the coupling of l-iodopyrene in refluxing
Results and
piperidine (Scheme 60, Conditions B) gave a yieldof
-----:>
corEt
AcN
I
H
AcN
63
70Vo.
CO2Et
I
H
127
126
Conditions
Conditions
Discussions
A: 70 (0.66 eq), Pd(PPh3)4 (0.1 eq), CuI (0.2 eq), EqN, DIvIF1'499o
B: 70 (0.66 eq), Pd(PPh3)4 (0.1 eq), CuI (0.2 eq), PP\ (0.2 eq)' piperidine;
517o
Scheme 60.
H
AcN
I
H
corEt
AcN
I
H
corEt
Í28
Catalytic hydrogenation of 127 proceeded in EtOAc overnight in94Vo yield (Scheme
61). Reduction to the saturated compoundl2g
was confirmed by MS
(M. 387),
the
tH NMR of 127 of the resonance at õ 3.25 (d,zIJ, J 4.7 Hz)
disappearance in the
correspondingto
1.7
CH'-G{ and the appearance
in the lH spectrum of 129 of resonances at
5-2.05 (m, 4H) and 3.26-3.42 (m, 2}I).
----->
H
AcN
I
H
corEt
H
AcN
I
H
L27
corEt
129
Í1", 5Vo Pd/C, EtOAc;94Vo
Scheme 61.
Results and Discussions 64
As homocoupled alkyne dimers may be formed to a significant extent in PdCC
reactions, the couplin g of 126 to 69 was attempted in order to synthesise the cross-coupled
producte6 130 (Scheme 62). Subsequent hydrogenation of 130 would then give a labelled
glycine derivative which incorporates a longer spacer unit. However TLC of the mixture
after reaction under standard conditions at room temperature overnight showed spots for
unreacted 69, alkyne dimer 114 and (presumably) acid dimer 128; no other fluorescent spot
for 130 was found. This suggested that the kinetic
acidityeT
of the alkyne proton on 127 was
probably much grcater than for 69 and that for successful cross-couplinges of the alkynes
activation of one of the alkynes as a halogen or stannaneee derivative would be necessary. As
it
appeared unlikely that other labels would give a different result, experimentation
wlthl2T
was concluded.
corEt
AcN
I
H
127
->
AcN
I
CO2Et
H
130
69 (1 eq), Pd(PPh3)4 (0.1 eq), CuI (0.2 eq), EtrN, DMF.
Scheme 62.
It
has been shown that the palladium catalysed cross-coupling reaction of terminal
alkynes and aryl iodides/triflates is an efficient method for the prepalation of labelled
aromatic amino acid derivatives (except for the tryptophan derivative 113). Also, the
pyrene-glycine derivative 127 was prepared in good yield using this methodology.
Fluorescence was not quenced upon coupling of the labels to the amino acids; a comparison
of the fluorescein label 84 and the corresponding phenylalanine adduct 122 is shown in
Figure 4. Such labelled amino acids (suitably protected) should be able to be used in the
construction of modified small synthetic peptides (using both automated and manual
methods) which display the advantages previously mentioned (InÛoduction, p 18).
Results and
Discussions
65
9û
t,
i1
f
'Ìtr
1
I
I
60
I
I
50
oá relative
1
1
40
intensity
30
1'.
I
ti
20
i¿
l0
0
500 nm
600
(a) Compound 84 (c = 5.7 x
10-6
mol dm3)
(:\
.r:a
.: ì
::i
atl:
80
?0
t
60
I
o/o
relative
intensity
t
I
f
.t
1
1
1
I
0
nrn
600
(b) Compound,l22 (c = 6.9 x
10-6
mol dm-3)
Fluorescence Spectra of Compounds 84 andL22 (ethanol solutions, f,", = 483 nm).
Figure 4.
Results and
Discussions 66
Chapter 3.2. Coupling of Label-spacer Molecules to Nucleoside
Derivatives
The palladium catalysed cross coupling reaction between 5-halo or S-triflyluridine
derivatives and terminal alkynes has been used to prepare S-alkynyl substituted compounds,
which may have antiviral or anticancer activityst'ez
*¿
in the preparation of 5-propargylamine
adducts, which were reacted with fluorescent labels". Also, 8-alkynyl substituted adenosine
and guanosine derivatives have been synthesised from the respective 8-bromo derivatives,
which has allowed the preparation of compounds which have been æsted for cytokinin
activityes and A, adenosine receptor activity52.
The reactions generally occured in high yields and under mild conditions; protection
of the sugar hydroxyl groups, although not necessary for the coupling reaction, was generally
used for chromatographic and characterisation purposes. Hence the nucleoside derivatives
51, 52 and 53 selected
for coupling reactions were protected as the di- or triacetate by
reacrion with acetic anhydride in pyridine (Scheme 63) to give the protected compounds 131,
132 and133 respectively. The melting points of the products were comparable with the
literature, and FABMS,
rH and t3C spectroscopy were consistent with the proposed structures.
AcO
HO
--->
R
2eq&crO 131: R=H
51: R=H
132: R = OAc
3 eq Acp
Base = 8-Bromoadenine 52: R = OH
133: R = OAc
3 eq AcrO
Base = 8-Bromoguanosine 53: R = OH
Scheme 63. AcrO (2 ot 3 eq), pyridine, 0o to RT overnight.
Base=S-Iodouracil
The reaction of protected 5-iododeoxyuridine 131 with the biotin label 107 was
considered
first. The production of cyclised isomer 135 (which previous reports have
indicated is catalysed by copper saltssto) is
a
potential problem, however reaction in DMF has
been shown to reduce the amount of cyclised isomer formedlm. A mole ratio of 2:1 copper to
Results and
Discussions 67
pallad.ium has been shown to offer best coupling conditions with the minimal production of
sideproducts3l". IIence the initial rcaction conditions used for the couping of the biotin label
107 are shown in Scheme 64 (Conditions
A). TLC (10/90 MeOIVCff"Or) of the da¡k brown
reaction mixture after stirring at room temperture for 3 hours showed the absence of 131 at
\
0.64, and a new fluorescent spot (î'". =365 nm) at & 0'20' After workup and
chromatography,
tH NMR showed the expected resonances for the productl34; also, an
unexpected low intensity resonance at ô 8.19 was observed. The resonance was assigned to
the vinylic proton in the furan ring of the cyclised isomer 135, and integration relative to C4
showed a ratio of 134 to 135 of approximately 4:1. Repeated column chromatog¡aphy was
unable to effect separation, and as reducing the amount of base from 2.5 to 1.2 eq has been
shown to reduced the amount of cyclisation producte2 , the reaction was repeated under these
modified conditions (Scheme 64, Conditions B). Interestingly, the reaction mixture turned
a
light yellow colour after the addition of the Pd(PPh3)4, and remained a light colour throughout
the reaction. The adduct 134 was recovered in86Vo yield, and no trace of fluorescent 135
was observed; by TLC, the spot corresponding to 134 was no longer fluorescent. Synthesis
lH and t'C spectra were consistent with the
was confirmed by FABMS (M+H.704); the
proposed structure. Similarly, reaction of 131 with the fluorescein label SL (Scheme 65)
gave the adduct 136 in 767o yield, and there was no evidence for the formation
of any
tH and r3C
cyclised byproduct. Coupling was confirmed by FABMS (M+H. 807), and the
NMR spectra were consistent with the proposed structure.
Results and
I
HN
Díscussíons 68
H
HN
N
AcO
--+
^
AcO
o
131
Conditions
Conditions
A:
B:
L34
107 1.5 eq, Pd(PPh3)4 (0.1 eq), CuI (0.2 eq), EqN (2.5 eq),DMF, RT; 567o'
107 1.2 eq,Pd(PPq)4 (0.1 eq), CuI (0.2 eq), EgN (I.2 eq), DMF, 40": 86Vo.
Scheme 64.
o
il
c(cHt4
¿
H
H
N
H-N
N
Yo
N -H
AcO
AcO
135
o
I
HN
HN
________>
131
SL
(l.zeq),
o
136
Pd(PPh3)4 (0.1 eq),
CuI (0.2 eq), EtrN (1.2 eq), DMF, 40'; 76Vo.
Scheme 65.
Results and
Discussions 69
Coupting of protected 8-bromoadenosine 132 with the acridone label69 (Scheme 66)
was attempted next. TLC (EtOAc) of the reaction mixture afær 24 hours stirring at room
temperature showed the absence of 132 at Rr 0.42, a small amount of label at
new spot corresponding to product at
in
897o
& 0.38. After workup the product
&
0.78 and a
137 was recovered
tH and t'C NMR spectra
yield; coupling was confirmed by LSIMS (M+H. 737), and
were consistent with the expected structure. Simitarly, reaction of 132 with the biotin label
107 under the same conditions (Scheme 67) gave the biotin adduct 138 in 887o yield.
lH and t'C NMR spectra wereSynthesis was conf,rmed by LSIMS (M+H.785), and
consistent with the expected structttres.
Br
€
AcO
AcO
AcO
OAc
OAc
I37
132
(2.5
RT,z4ht:897o(0.2
DMF,
eq),
EqN
(0.1
eq),
eq), CuI
69 (1.5 eq), Pd(PPh3)4
Scheme 66.
o
N
Br
lt
---------:>
H
AcO
AcO
AcO
OAc
AcO
N-H
H-N
OAc
132
107 (1.2 eq), Pd(PPh3)4 (0.1 eq), CuI (0.2 eq), EqN (eq),
Scheme 67.
o
138
DMF, 50", ON:
887o.
Results and Discussions 70
Guanosine derivative 133 was reacted with the fluorescein label Sl under standard
conditions (Scheme 68). After 5 hours stiring, TLC (10/90
MeOfVCtlC!)
showed yellow
fluorescent spots at Rr 0.68, O.27 and0.17 corresponding to the label, homocoupled label
dimer and product respectively, and the absence of a spot atRy0.22 corresponding to 133.
After removal of solvent, chromatographic separation of the residue gave the fluorescein
dimer 140 in I2Vo yield (based on starting label), followed by the coupled product 139, which
was impure. A large amount of low
\
material was observed on the column, which failed to
elute with a higher polarity solvent mixture. Upon repeating the chromatography, 139 was
obtained in 5l7o yietd as a bright orange glass. The low yield may be due to the partial
complexation of the metal catalysts. Any such complex formed would be charged and hence
presumably would bind strongly to silica; this may account for the majority of the mass
lH and r3C
balance of starting materials. Synthesis was confirmed by LSIMS (M+H.904);
spectra were consistent with the coupled product
139. Similarly, reaction of 133 with the
biotin label 107 under the previous conditions (Scheme 69) and workup gave the labelled
tH and
compound 141 in 587o yie\d. Synthesis was confirmed by LSIMS (M+H. 801) and
t'c NMR.
Br
HzN
HN
HzN
N
-=--J>
AcO
OAc
AcO
Ac
139
133
S1 (1.5 eq),
Pd(PP\)4 (0.1 eq), CuI (0.2 eq), EqN (eq), DMF, 50', 5 hours:
Scheme 68.
517o.
Results and
Díscussiorls 7l
140
HN
Br
HN
(CHz)¿
HzN
HzN
-------:>
H
H
H
Yo
H
l4l
133
107 (1.5 eq), Pd(PPh3)4 (0.1 eq), CuI (0.2 eq), Et N (eq), DMF,50", 4 hours: 587o.
Scheme 69.
The results of these coupling reactions show that large label-spacer adducts may be
coupled easily and in very good yields for the 5-iododeoxyuridine derivative 131 and
8-bromoadenosine derivative 132; however the 8-bromoguanosine derivative 133 couples in
only average yields. The preparation of labelled deoxynucleosides for incorporation into
oligonucleotides via standard solid phase methodology should easily be achieved. This
methodology may also be applicable to preparation of oligonucleotides which are
functionalised with intercalating moities from a nucleobase2o.
Results and
Discussions 72
Chapter 3.3. Coupling of Label-spacer Molecules to Steroid
Derivatives
Steroids with carbonyl groups such as 142 and143 are easily converted to triflate
derivativeslot and have been shown to undergo a variety of palladium catalysed processesto2;
the cross coupling with terminal alkynes has been well investigatedas'ss. As labelled steroidal
derivatives have been used as probes in biological systems3t'to' (particularly in the study of
membrane structures"'"¡, the possibility of coupling estrone triflate derivative 54 and
epiandrosterone triflate derivative 55 with the developed pyrene labels 74 and 78, and biotin
label 107 was investigated.
cH3o
t42
L43
Reaction of 54 with pyrene label74 under standard conditions (Scheme 70)
proceeded readily. TLC (20lS0 CHrcl/hexanes) of the reaction mixture after 3 hours stirring
at room temperature showed a now fluorescent spot at
&
0.31 corresponding to product, and
the absence of the triflate at Rr 0.10. After workup and chromato$aphy, the product 144 was
recovered in927o yield. MS showed a molecular ion
atmlz6l6 and tH
and
t'C NMR
spectra were consistent with the expected structure.
_-______>
MeO
54
L44
Conditions:74 (1.2 eq), Pd(PPh3)4 (0.1 eq), CuI (0.2 eq), Et N,DMF,3 hours RT;927o.
Scheme 70.
Results and
Discussiots 73
Similarly, reaction of 54 with biotin label 107 under standard conditions (Scheme 71)
gave the labelled product 145 in 897o yield. The reaction occurred in less than three hours, as
tH, t'C NMR)
indicated by the absence of starting triflate by TLC, and the spectral data (MS,
were in agreemont with the expected structure.
o
il
€
H
H-N
-H
MeO
54
145
Conditions: L07 (L.Zeq), Pd(PPhJ4 (0.1 eq), CuI (0.2 eq), Et N,DMF,4 hours RT; 897o.
Scheme 71.
Reaction of androsterone derivative 55 with pyrene label 78 under standard conditions
(Scheme 72) to give 146 occurred in857o yield, and the spectral data were consistent with
the proposed structure. Repeating the reaction with the biotin label 107 (Scheme 73) gave
the adduct
in
887o
yield; again the spectral data were consistent with the expected product.
Ac
_______>
Tfc
55
146
Conditions:78 (1.2 eq), Pd(PPq)4 (0.1 eq), CuI (0.2 eq), Et N, DMF, 4 hours RT; 857o.
Scheme 72.
Results and Discussions 74
--------->
1
TflC
(cHt4cN
H
=H
N H
H
147
55
Conditions:107 (1.2 eq), Pd(PP\)4 (0.1 eq), CuI (0.2 eq), Et N, DMF, 4 hours RT; 887o.
Scheme 73.
These results showed that the selected steroidal triflates may be labelled readily and
in
high yield using PdCC methodology. Other steroidal triflates have been shown to have
similar reactivityas55'lo2, and so this may be a general method for the labelling of steroids
which are functionalised with
a
carbonyl group. As a hydrocarbon spacer unit can be
incorporated with the label, the lipophilicity of adducts may be enhanced, which could prove
advantageous in some systems.
Results and
Discussions
15
Chapter 4. Gelation of Organic Solvents by Biotin Amides and
Esters.
Gelation is
a
well known phenomenont*. It occurs when
a
liquid displays "solid-like"
behaviour due to the presence of a smalt amount of solid forming a network which
incorporates and retains the liquid (which is the major component). Defining a gel is difficult
due to the diverse nature of systems which display gel-like behaviour, however the following
criteria include almost all systems which a¡e currently catagorised as gelsl6: (i) A gel consists
of a three-dimensional network composed of basic elements connected in some way and
swollen by a solvent; (ü) Gel formaúon or gel melting should proceed via a fust order
transition (which implies there exists a well-defined temperature (the gelation temperature,
\)
below which the solute-solvent mixture has solid behaviour; (iü) A gel immersed in an excess
of preparation solvent should be unaffected or swell but not dissolve or disaggregate; (iv) A
gel is a system which can be removed from the vessel in which it has been prepared without
losing its shape or integrity.
The molecules of the network which entrap the solvent may be connected by covalent
bonds (chemical gels) or by hydrogen bonding and van der'Waals interactions (physícal gels).
When chemical gels are heated to the point that the covalent bonds break, irreversible
degredation impedes the reformation of a similar system. In conEast, heating a physical gel
disrupts the secondary bonds which can reform upon cooling, thus physical gels are also called
thermoreversíble gels. Physical gels of gelatin or other biopolymers in aqueous solution have
been known for a long time, and
it was believed that gelation occurred only for such
systems.
However the fust thermoreversible gels formed in the organic solvents 2-butanone and
cyclohexanone by poly(vinylchloride) polymers rvere reported in 194716. Recently the gelation
of organic solvents by low molecular weight compounds has been an active a¡ea of
investigation. The rccovery of spilled solvents, disposal of used cooking oil and use in drug
delivery systemslørot have been suggested as possible applications. About 12 groups of
compounds (not including biotin derivatives) are known to gelate organic solventslffi. They
can be divided into two classes; compounds which associate via hydrogen bonding and van der
Results and
Discussions 76
Waal interactions to form gelsl@Jto'ttt (e.g. 149), and compounds which form gels via van der
tz't tr't ta
V/aal interactions onlyl
(e.
g. 107).
CHMe
(cH2)ecH3
-H
(cHrecH3
149
148
o
H
N
H
S
H
107
The observation that the attempted recrystallisation of biotin-N-(l1-undec-1-yn)-amide
(107) from EtOAc gave rise to a gel was unexpected. Formation of the gel was achieved by
heating 107 (about 150mg) in boiling EtOAc (about 35
ml). As the compound dissolved,
the
solution thickened markedly and upon being allowed to cool to room temperature gelled. The
gel was transparent, stable to mechanical inversion and retained shape upon lifting with a
spatula, although vigorous treatment caused degradation of the gel to solvent and crystals of
107. Reheating the mixture reformed the gel, i.e. it was thermoreversible. Storage in
a sealed
vial prolonged the life of the gel; upon removal of the seal solvent was slowly lost, the gel
shrank and a xerogel was formed. The gel did not show any structure when viewed under an
optical microscope, and both the gel and xerogel did not transmit light when placed between
crossed polarising filters (i.e. were not birefringenÐ. Numerous other solvents were tested
for
gelation, and the results are summarized in Table 3. Toluene formed the most mechanically
stable gel, and at the lowest concentration. The gel was unchanged even after it was left
Results and
Discussions
77
uncovered in a fume cupboard for 30 days. Subsequent mechanical agitation of the gel
resulted in the solvent being lost overnight.
Table
3. Concentration
and Molar gelation ratios
of 107 with several solvents.
Concentration
(gdm")
Molar ratio
4.2
960
Benzene
3
1,470
Ioluene
3.2
1,040
Chloroform
L2
410
Solvent
Ethyl acetate
tH NMR spectroscopic analysis of a series of increasing concentrations of 107 in
CDCI3 showed the chemical shifts of the amide protons moved markedly downfield, consistent
with aggregation which may lead to formation of a hydrogen bonded network (Iable 4). The
spectra were relatively well resolved (Figure 5a) although as the concentration increased
resonances were broadened slightly, and smaller poorly resolved resonances became apparcnt
(Figure 5b), presumably due to the network
.
As the solvent was gelled at these
concentrations, it was anticipated that a loss of resolution would have occurredrrs. The
apparent resolution may be explained by considering that 107 exists in two distinct statesttt.
The first is as monomeric or intermolecularly hydrogen bonded species in solution which rotate
quickly enough to exhibit well resolved resonances, and the second is in the hydrogen bonded
network whose resonances are broadened due to their restricted rotation. The specEa at
higher concentrations are superpositions of the two states. Molecular modelling studies
suggest that the energy minimised structure of a possible dimeric species, which is shown
in
Figure 6, has strong intermolecular and inramolecular hydrogen bonds, with an
oxygen-hydrogen distance of about 2.1 Angstroms.
The relatively well resolved spectra in CDCI, is in contrast to the spectrum of a
[2Hr]-toluene gel which was poorly resolved at room temperature (Figure 7a). Increasing the
tempemture of the sample in 20" steps showed the resolution improved betweeen 80' and 100",
presumably when the gelation temperature
(\",)
was exceeded (Figure
7b). Increasing the
Results and
Discussions 78
temperature of a 10mg/0.6m1 gel in CDCI, showed large upfield changes in the chemical shifts
(Table 5) and broadening of the resonances for the amide protons, consistent with disruption
in the hydrogen bonding which exists between the presumed dimeric, trimeric etc. associations
of molecules.
Table
4
Chemical shift of amide protons of 107 vs concentration.
Concentration"
N1'-H
N3'-H
cHrN¡/CO
0.5mg
4.56
5.t2
5.45
2.0mg
4.85
5.57
5.59
5.0mgb
5.13
5.95
5.73
10.0mgb
5.31
6.16
5.82
25.Omgb
5.3s
6.r9
5.84
(notes: þer 0.6m1 CDCI.; bgel formed)
Tabte
5
Chemical shift of amide protons of 107 (ô ppm) vs Temperature (K)
T (K)
Nl.'-H
N3'.H
cH"NrlCO
293
5.62
6.23
5.91
303
5.23
6.01
5.82
3t3
5.09
5.79
5.71
323
4.97
5.61
5.61
Figure 6. Possible Dimer formed by Biotin Amide derivatives.
lSide chain hydrogens (apart from amide proton) removed for clarity.]
Results and
6.5
6,0
5.t
5.0
a.5
3.0
3.5
a-0
PPI
?.6
Discussíons 79
2.0
t.õ
1.0
(b) 25mg / 0.6 ml
5.0
4.5
,a.0
3.5
PPX
3,0
2.5
2.0
!.5
(a) 0.5mg/0.6m1
Figure 5. lH NMR spectrum of compound 107 in CDCI, gel at 293K
Results and
6.5
6.0
¡.6
5-5
PPX
1.O
3.5
3.0
2.5
Discussions 80
t.5
2-O
(b) 373 K
7.0
6.5
6.0
õ-l
1.0
il.6
4.0
PPII
(a)
5
3.0
2.é
2.O
1.5
t.0
293K
2¡H1r-toluene (10 mg 0.6 ml)
/
Figure 7. lH NMR of compound 107 in
t.0
Results and
Discussions
81
Urea derivatives a¡e well known hydrogen bond donor and acceptor moietieslrT, and
have ofæn been used for molecular recognition purposestt*. The self assembly
of
five-membered cyclic urea derivatives, in which the amide protons are constrained syz to the
carbonyl group is not surprising given their symmeuical nature. Possibly an extendeS linear
network ( a "ribbon") composed of ttre 2-oxoimidazole rings intermolecularly hydrogen
bonded is formed. A model on the molecular scale is shown in Figure 8, and energy
minimised molecular modelling suggests the hydrogen bonding distance to be about 2.1
Angstroms. As gelation has been proposed to be a form of incomplete crystallisationlle,
iregularity in the network wilt help stabilise the gel state. This is achieved
as the
tetrahydrothiophene ring may be incorporated above or below the plane of the hydrogen
bonded network. The long alkyl chain solubilises the molecule in organic solvents, and
enhances the gelation process by its conformational
flexibility.
Figure 8. Possible hydrogen bonding network formed by the bicyclic rings of biotin.
The possible uses of compounds which gelate organic solvents may necessitate the
inclusion of low polarity solvents such as aliphatic hydrocarbons. The amide 107 was not
soluble in hexane, howeve¡ it was realised that a more lipophilic analogue may be and could
possibly form gels. The most obvious modifications would be to either lengthen the alkyl chain
of the amide, connect the alkyl moiety via an ester, or both. Hence a series of biotin amides
and biotin esters with varying alkyl chain lengths (n-propyl, n-hexyl, n-octyl, z-undecyl,
Results and
Discussions
82
n-dodecyl, n-hexadecyl) were synthesised in order to determine the scope of gelation of
solvenrs by biotin derivatives. Reaction of biotin NHS ester 106
witlt the amine in DMF
(Scheme 74) gave the amides in good yields. The reactions were generally over (as indicated
by absence of 106 by TLC) within 2 hours. Removal of the precipitated NHS by filtration,
removal of the solvent in vacua, chromatography and recrystallisation gave the amides
as
colourless amorphous solids. Biotin esters were synthesised by reaction of biotin (41) with 5
equivalents of alcohol in refluxing toluene, catalysed byp-toluenesulphonic acid (Scheme 75).
Heating at reflux for 48 hours, removal of the solvent in vacuo, filfation to lemove uireacted
biotin, and chromatography gave the esters in poor to excellent yields. The propyl ester 161
was prepared in a similar manner, however the reaction solvent was propan-1-ol and the period
of reflux was 6 hours (Scheme 76). The undec-10-yn-1-ol ester 162 was prepÍLred by reaction
of 106 with the alcohol 59 in DMF, catalysed by DMAP (Scheme 77). The products were
recovered after chromatography generally as colourless amorphous solids. Gelation tests were
performed by placing a weighed amount of compound (15-20mg) in
a
preweighed vial and
heating with the solvent to be tested. After dissolution (if not insoluble) and cooling at room
temperature for 60 minutes the outcome of the experiment was noted. A gel was considered
to have formed
if
a
transparent mass which did not flow upon inversion of the vial resulted.
The total mass of the vial and solvent was determined and the concentration of gelator
calculated. Gelation was generally complete by the time room temperature was reached; more
vigorous attempts to achieve gelation such as cooling the gel in cold water or a refrigeratorll3b
were not attempted. If the compound was soluble at room temperature, the amount of solvent
was reduced by evaporation and the concentrated solution studied for gelation as above. The
results of the gelation tests are shown in Table 6 for the amides and Table 7 for the esters.
Results and
Discussions
OH
\{(cH)ncHr
H
_______*
N
N
H
H
83
H
(Ð l,[HS (1 eq), DCC (1.1 eq),
DMF, 80" to RT, 2 hours. (iÐ CH3(CF!)'M!.
Scheme 74.
n
fuJdVo
150
151
152
153
154
155
15
11
10
7
5
2
83
7l
56
67
66
73
o
H
\
H
H
s
S
---------->
H
H
C4(CFL)"OH (5 eq),p-TsOH (0.1 eq), toluene, reflux 48 hours'
Scheme 75.
n
11
38
80
79
98
158
159
160
10
7
5
81
H
H
CH¡
o<
N
-------:)
4l
fuld7o
156
157
15
N
H
CH3(CH)pH, p-TsOH
H
161
(0. 1 eq),
Scheme 76.
reflux 6 hoats, 627o.
Results and
Discussions
NHS
84
o(cH2)e
S
N
H
H
N
->
H
L62
4L
Compound 59, DMAP (1 eq), DlvlF,467o.
Scheme 77.
The results of the gelation tests showed (i) the optimal length of the alkyl substituent
is between 8 and 12 carbons (n-octyl to n-dodecyl), i.e. an overall (including pentanoic chain
of biotin and heteroatom) length of 14 to 18 atoms; (ii) esters a¡e less polar than amides, and
the n-dodecyl, n-undecyl and n-octyl esters (157, 158 and 159 respectively) gel hexane when
predissolved in CHrCt; (iii) the octyl ester 158 gelled light paraffin oil when predissolved in
CÍlrcl,however crystallisation occurred after
14 days;
(iv)
a terminal alkyne is important
in
enhancing the gelation process. Terminal alkynes are known to undergo weak hydrogen
bond.ingl2o, however the chemical shift
of the acetylenic proton showed no significant change
in either the variable concentration or variable temperature experiments. How the alkyne
enhances the gelation process is unclear. As the undecylamide is not such an efficient gelator
this shows how a smalt difference in structure leads to a large difference in gelator behaviour.
Further work is required to determine the structure of the hydrogen bonded network
on both the molecular and macroscopic scales for this new class of thermoreversible gelator
compounds. The compounds a¡e readily synthesised, gel at low concentrations, may form
very stable gels and can be designed to gel hexane. Analogues incorporating branched alkyl
chains, dialkylamines, terminal alkynes and/or various alkyl lengths or other features which
may give rise to enhanced gelation should be easily synthesised.
Results and
Table
6: Gelation of Solvents by Biotin
Discussíons
85
Amide Derivatives
Solvent
(16)
(r2)
(1 1)
(8)
(6)
cc14
s
c
c
c
1
cHc13
s
18
2l
22
c
c
ürrcl"
I
c
c
c
I
I
I
ETOAC
I
c
c
c
c
4.2
hexanes
I
i
c
I
I
1
1
I
toluene
s
c
c
c
c
I
3.2
acetonitrile
c
c
c
c
c
c
c
ether
S
1
L
1
I
I
1
MeOH
c
c
c
s
s
c
c
aceton0
I
c
c
c
c
c
c
(undecyn)
(3)
i
I
'12
3
benzene
(i insoluble, c crystallise, s soluble, number = conc (gdm') of gel)
Table 7: Gelation of Organic Solvents by Biotin Esters
Solvents
(16)
(t2)
(1 1)
(8)
(6)
(3)
undecyn
cc14
c
s
s
43
9
L7
s
cHC13
s
s
s
s
S
s
s
s
s
s
S
s
s
s
EtOAc
c
s
s
s
S
s
S
hexanes
1
c
c
1
1
1
1
toluene
c
s
S
s
s
s
s
acetoniüile
c
c
s
s
s
s
s
ether
I
c
1
I
1
I
1
MeOH
s
s
s
S
s
s
s
acetone
c
s
s
s
s
s
s
c
,k
gl2c\
cFI,Cl,lhex
c
5.3
paraffin
*formed
c
9.9
c
2.4
a transparent
polymeric mass
(i insoluble, c crystallise, s soluble, number = conc (gdm') of gel)
86
Chapter 5. Summary
The synthesis of novel label-spacer molecules in which a hydrocarbon chain is
attached to a reporter moiety (acridone, biotin, dansyl, fluorescein, pyrene, and
tris(l,10-phenanthroline)ruthenium) at one terminus, and is functionalised at the other
terminus with an alkyne, has been achieved. The palladium catalysed cross coupling reaction
between aryVvinyl halides/triflates and terminal alþnes has proved to be an efficient method
for the introduction of the label-spacer molecules to halogenated and triflated derivaúves of
biomolecules (amino acids (apart from ûyptophan), nucleosides and steroids).
The biotin label 107 was found to gelate benzene, chloroform, ethyl acetate and
toluene. Variable temperature
tH NMR spectroscopy of gels in CDCI, and 2¡H1r-toluene
suggested the formation of a polymeric hydrogen bonded network, presumably formed by the
cyclic urea moiety of 107, which entraps solvent. Some n-alkyl biotin ester and amide
derivatives were found to form gels in hexane and paraff,rn oil at low concenEations.
Experímental
87
Experimental
All reactions were routinely performed in oven dried glassware under a nitrogen
aünosphere (unless in aqeuous solution), and palladium catalysed cross coupling reactions
were performed in Schlenk glassware. Melting points were recorded on a Reichhert hot stage
apparatus and are uncorrected. Proton and Carbon NMR spectra were recorded on a Bruker
ACP-300 or a Va¡ian Gemini 200 spectrometer. CDCL was used as a solvent unless
otherwise stated, with tetramethylsilane used as an internal standard. Mass spectra were
recorded on VG ZAB
2Iß
mass spectrometer with either electron impact (EI) or fast atom
bombardment (FAB) ionisation, or on an AEI-GEC MS 3074 instrument with EI ionisation.
Accurate mass determinations using EI or Liquid Secondary Ion MS (LSIMS) were made by
the Organic Mass Specrometry Facility at the University of Tasmania, or using EI at the
Department of Chemistry, University of Melbourne. Ultraviolet spectra were recorded on a
Pye Unicam SP8-100 spectrometer. Fluorescence spectra were recorded on a Perkin Elmer
3000 spectrometer. IR spectra were recorded on a Hitachi 270-30 spectrometer and data
processor.
Triethylamine, pyrrolidine, piperidine and
nitrogen and stored over
4,Ä,
Cflcl
were distilled from
CaI! under
molecular sieves. DMF was distilled from CaII, (ca. 80'at
2gmmHg) and stored over 4Å molecular sieves. EgO and TTIF were freshly distilled from
sodium and benzphenone under nitrogen. Methanol was fractionally distilled under nitrogen
and stored over 3Å molecular sieves. Other regents were purified according to according to
literature procedurest2t.
Analytical thin layer chromatography was carried out using Merck aluminium sheets
precoated with kieselgel 60
Fzs¿
or (when stated) with alumina 150 Frro, and visualised using
either a254nm or 365 nm lamp, or with a4Vo solution of phosphomolybdic acid in ethanol.
Flash chromatography"'was caried using Merck kieselgel 60 (230-400 mesh) or (when
stated) on alumina UG, and solvents used were distilled before use.
Compounds synthesised according to literature procedures: 1-bromopyrenet",
1-iodopyrenetu, N-phenyltriflimidelã, åis-1,10-phenanthrolineruthenium(tr)dichloridel26,
Experímental
88
tetrakis(triphenylphosphine)palladiumr2t, estrone triflatelOà, androsterone triflatel0h. A
sample of dimethyl-(1-diazo-2-oxopropyl)phosphonates6 was a gift from Mr. C. de Savi.
The following abbreviations have been used in defining peak shape for the various
spectra:
tH and ttc NMR; b
=broad, d= doublet,m= multiplet, q = quartet, s = Singlet,
triplet. IR: å = broad,
rn = mediurr, s = strong,
w= weak; UV andfluorescence
t=
spectra; s/r
rH NMR. The numbering on structures is for
= shoulder. Unless otherwise stated J =3J for
NMR resonance assignment only. Mass spectral data are rcported: mlzratio
(Vo
relative
abundance). UV data are reported: î,-", (e). Fluorescence data are reported: À-"* (7o
relatle
intensity).
Due to the lack of reproducibility with microanalytical data the labelled biomolecules
were characterised by
tH and r3C NMR, with high resolution mass spectroscopy @I and
LSIMS) confirming the molecular formulae.
Experimental Described in Chapter 2.1.
Undec-l0-ynoic acid (57)
Redistilled lQ-undecenoic acid (1089, 0.59 mol) was dissolved in dry CClo (300m1) and
cooled in an ice/methanol bath to approximately -10". Bromine (969, 31m1, 0.60 mol) was
added dropwise over 90 minutes, the cooling bath removed, the reaction mixture allowed to
come to room temperature and the solvent removed. A solution
of KOH (2729,4.85 mol) in
water (160m1) was added and the mixture stired at 125" (oil bath temperature) for
t
hours.
After cooling to room temperature, the mixture was poured into water (1500m1), acidified to
pH
1
with IOToH"SOo(approximately
500m1) and extracted
with CÍlrcl, (4 x 250m1). The
organic extracts were combined, washed with saturated brine (500m1), dried (NarSO.) and the
solvent removed. The residue was distilled through a 150mm Vigreaux column, the fraction
boiling between I2E-136" at 0.02mmHg being collected. Recrystallisation from hexanes gave
the title compound as colourless crystals mp 43" 0it56.
43) in 18.1g (I7Vo) yield.
Experimental
89
IR (film): 3350-2500br (O-H),3300s (=C-H), 2L40w (C=C), l72}s (C=O). 'H NMR:
1.33-1.67 , m, l6IH, methylene protons;
IJz, CH"-G);2.35 (t,2]H, J 7 .3
lgrz,
I.94 (t,lH,
uf
2.6H2, =C-H); 2.1.8 (dt,2H, J 7 .O,2.6
l'C NMR: 18.8,
CI{r-COrH); 9.95 (ås, lH, COOH).
25.1,29.1,29.2,29.3,29.4,29.6,34.6 (alkyl),68.6, 85.2 (alkynyl)' 180.7 (CO2H).
Undec-10-yn-1-ol (59)
A solution of undec-lO-ynoic acid (57) (12.09,66 mmol) in EqO
(100m1) was added
dropwise to a stirred suspension of LiAlHo (2.509, 66 mmol) in EqO (150m1). The mixture
was heated to reflux overnight. After cooling to room temperature, the reaction mixture was
hydrolysed by the dropwise addition of water (2.7m1),157o NaOH (2.7m1) and water (2.7m1).
Afte¡ filtration to remove the lithium salts, the solvent was romoved and the residue distilled,
bp 75-76" ar 0.02mmHg
(1it128.
111-112" at 4mmHg) to give the title compound as a
colourless oil in 9.609 (87Vo) yield. IR (film): 3400-3100år (O-H), 3304s
2852s,2L10w
1.93 (t, lld,
(H-G), 2924s,
(GC), 1466m,1058s. tH NMR: I.29-1.59, (m,l4IJ, methylene
f 2.6}jr2,{-H);
protons);
2.L7 (dt,2IJ, J 7.0,2.6H2, CH"-G);3.62 (t,2IJ, J 6-6H2,
cHr-oH). t'c NMR: 20.4,27.7,30.4,30.7,31.0,31.3, 3r.4,3L.5,34.7 (akyl);70.r,86-7
(alkynyl).
Undec- 10- yn-L-O - p -toluenesulphonate (60)
To a stined solution ofp-toluenesulphonyl chloride (11.339, 59.4mmo1,2 eq) in pyridine (75
ml) at 0' (icebath), was added dropwise a solution of the alcohol 59 (5.009, 29.7 mmol) in
pyridine (25 ml) over 60 minutes. Stirring was continued as the ice bath warmed to room
temperature overnight, then the reaction mixture was poured into water (100m1) and extracted
with EqO (4 x 50ml). The organic extracts were combined, washed with LÙVoHCl (50m1),
water (50m1), dried (NqSO) and the solvent removed to give the crude product in9.02g
(94Vo)
yield. The crude product was converted immediately to the iodide
Undec-10-yn-1-O-methanesulphonate (61): Method A.
as described below.
Experimental 90
Alcohol5g (5.009, 29.7 mmol) was dissolved in pyridine (20m1) and cooled in an ice bath.
Methanesutphonyt chloride (3.4Og,29.7 mmol) was added dropwise over 30 mins and the
mixture stired for 4 hours. The mixture was poured into water (100m1), the organic layer
separared, diluted
with CtlCl, (50m1), and washed successively with l07o
aQueous
HCI (3 x
50ml), water (50m1), brine (50m1), dried over NqSOo and the solvent was Íemoved to give
thecrudetitlecompoundin 6.829(937o)yield. IR(frlm): 3292s (H-C=)' 2928s,2852s,
2Il6w(GC), I468m,I354s,ll76s. tHNMR:
(quintet,
2IH,
1.30-1.41 (m,l{H,methyleneprotons);1.75
J 7 .2Hz,CH2-CH2-OMs); 1.95 (r' LH' J 2.6 Hz':C-H); 2.19 (dt, 2H, J 6.9, 2.6
IJz, CH"-É); 3.01 (s, 3H, CHr-SOr); 4.22 (t,2H, J 6.6 }Jz, CF!-OMs.)
Undec-10-yn-1-O-methanesulphonate (61): Method B.
To a stirred mixture of 59 (5.009, 30 mmol) and EgN (3.019, 30 mmol,
(50m1) was added dropwise methanesulphonyl chloride (3.4tg,3Ommol,
1 eq)
in
Cflcl
I eq). The mixture
was stirred at room temperature for 24 hours, filtered to remove precipitated
triethylammonium chloride, washed with water (50m1), dried over MgSOo and the solvent
removed. The crude product was recovered as a pale yellow oIIin 6.429 (88Vo) yield, with
tH and IR spectral data as before.
1
1-Iodoundec-1-ynel'e (62).
The crude tosylate (7.709,24 mmol) or mesylate (6.009, 24 mmol) was dissolved in dry
MEK (100m1), anhydrous NaI (30.09, 200 mmol) was added, the mixture refluxed for 24
hours, filtered to remove inorganic salts and the solvent removed. The dark yellow residue
was purified by squat chromatography (eluant hexane) to give the title compound as a
colourless oil in 5.069 (757o) yield. HRMS Calculated for C,,H,rI: 278.O532. Found:
278.0539. MS: 278 (M*, 100), 151 (M.-
I,30). IR (thin film):
2116 (C=C),1464,1432,1220,1178, 720.
3300s (H-C=)' 2924,2852,
tH NMR: 1.25-1.61 (m,l2H, methylene
protons); 1.82 (quíntet, J 7.0Hl2,2}J.', CH2-CII2-I); 1.95 (t, J 2.6 Hz, H-C=); 2.I9 (dt, J 6.9,
2.6Id2, CrL-C=); 3.19 (t, J',\.0rI2,2Ig',
28.40, 28.59, 29.19,
29
CÍ\-D. "C NMR:
7.18 (CI{2-I), 18.32,28.37,
.22, 30.39, 33.46 (alkyl), 68.06, 84.5 0 (alkyne).
Experimental
91
N-(Undec-10-yn-1-yl)phthalimide (64): Method A.
4
)
5
-(CHie:
6
7
To a srirred solution of phthalimide (265mg, 1.80 mmol) in DMF (10m1) was added NaII
(60mg, 807o suspension in oil, 1.98 mmol, 1.leq). The mixture was stirred for 15 minutes
then l-iodoundec-10-yne (62) (500mg, 1.80 mmol) was added and stirring continued for 3
hours at 100'. After cooling to room temperature the mixture was poured into water (100m1),
the aqueous mixture extracted with
CF!C\(3 x 30ml),
the extracts combined, washed with
water (50m1), dried (NqSO) and solvent removed. Residual DMF was removed under
vacuum (oil pump). The residue was separated by flash chromatography, eluant 15/85
EtOAclhexanes to give the product as a colourless oil which slowly solidified to white
crystals mp 45-48" in22lmg (4LVo) yield. HRMS Calculated for
C,rIlrNOr: 297.1729.
Found: 297.1724. MS: 297 (M*,42),149 (100). IR: 3284s (H-C=),3056w (tu-H),2I20w
(c=c),
L772s and 1710s (imide
methylene protons);
I.5l
(quintet, 2}J, J
CfL-Cft-phthalimide); 1.95 (r,
3.68 (r, 2IJ, J 7 .3
IHlz,
c=o), I6l6m (Ar C=C). 'H NMR:
LH,
f
7
.l
H:z,
1.30-1.35
(m,l0H,
CHr-CIL-C=C); 1.67 (quintet, 2}J, J 7 .3 H:z,
2.6 IJz, H-C=); 2.17 (dt, zIJ, J 7 -I, 2.6 }tz, Cfl"-C=);
CF!-phthalimide);
7
.68-7
.7
4 (m, 2}J, C3-H and C4-H) ; 7 .82-7 .87 (m,
2IJ, C2-Hand C5-H). "C NMR: 18.38, 26.81,28.44,28.57,28.67,28.98,29-10,29.30,
38.05 (alkyl),68.06, 84.14 (alkynyt), 1.23.14,132.20,133.82 (aryl), 168.45 (carbonyl).
N-(Undec-10-yn-1-yl)phthalimide (64): Method B.
A mixture of potassium phthalimide (1.649, 8.85 mmol , I.2 eq),1-iodoundec-10-yne (62)
(2.059, 8.02 mmol), 18C-6 (O.21g,0.8 mmol, 0.1 eq) and toluene (13 ml) was stirred for24
hours at 90". After cooling to toom temperature the reaction mixture was poured into water
(25 ml), extracted with CFlClr(25mI), the organic extract washed with brine (25m1), dried
(NqSO) and solvent removed. The residue was separated by squat chromatography eluant
EtOAclhexanes 20180 to give the product in 0.959 (40Vo) yield. The spectral data were
identical to those in the previous method.
Experímental 92
N-(Undec-10-yn-1-yl)phthalimide (64): Method CA mixture of phthalimide (400mg,2.72 mmol), 1-iodoundec-10-yne (62) (0.907mg,3.26
mmol, l.2eq),anhydrous &CO, (564mg,4.14 mmol, 1.5 eq) and 2-butanone (20m1) was
stirred at reflux for 48 hours. After cooling to room temperature the rection mixture was
poured into water (50m1), the aqueous mixture extacted with CFICL (3 * 25ml), the organic
extracts combined, dried (NarSO) and solvent removed. The method of purification and the
specgal data were identical with those in Method
A.
The product was recovered in 614mg
(78Vo) yield.
Undec-10-ynyl-1-amine (66): Method A.
A Soxhlet apparatus charged with undec-lO-ynamide (68) (5.00g, 28 mmol)
and fitted to a
flask containing a suspension of LiAlHo (1.229,33 mmol) in EqO (200m1). The suspension
was refluxed and the amide extracted until dissolved (approximately 30 hours), then lithium
complexes decomposed by the sequential addition of water (1.2m1), 157o NaOH solution
(1.2m1) and water (3.6m1). The grey precipitate was removed by
filtration, the solvent
removed and the residue distilled bp70-72" at 0.10 mmHg to give the title compound as a
colourless oil in 3 .92g (85Vo) yield. The oil slowly solidified to give a colourles s solid mp
53-55'. HRMS Calculated for C,,f!rN (M+H.): 168.1752. Found: 168.1756. MS:
168
(M+H*, 26),138 (48), 110 (39), 96 (43),86 (100). IR (film): 3500-3100rn (N-H str), 3304s
(H-G), 2It6w (GC srr). tH NMR: I.2I-1.48 (m,14 H, methylene protons);
1.86 (r,
lH,.I
t3C
2.7 Hz,H-C=); 2.09 (dt,2]F^,f 6.9,2.7 lFrz,CÍtt-C=i);2.60 (t,2H,J 6.6H2, CI{r-NIIr).
NMR: 20.33,28.81, 30.4t,30.66,30.98,31.38, 35.81,
44.20 (alkyl); 70.02,86.64 (alkynyl)
Undec-10-ynyl-1-amine (66): Method B.
A mixture of N-(undec-10-yn-1-yl)phthalimide (64) (1.219,4.07 mmol), hydrazine hydrate
(0.519, 0.50m1, 10.1 mmol) and EIOH (30m1) was strirred at room temperatue fot 24 hours.
The thick white precipitate was dissolved by the addition of l07o HCI to pH<l, the mixture
srfured
for
15 minutes and precipitated phthalhydrazide filtered
to pH>12 with
lM NaOH,
the aqueous layer extracted with
off. The frltrant was adjusted
CtlC\(z * 25ml),
the extracts
Experimental
93
combined, dried (NqSO), solvent removed and the residue distilled to give the product in
0.439 (64Vo) yield. The physical data (tH, IR') were identical with those in the previous
method.
Undec-l0-ynoyl chloride (67)
A mixture of undec-l0-ynoic acid (57) (5.0g,27 mmol) and SOCI, (4.09' 2.5m1,34 mmol)
was refluxed for 60 minutes, allowed to cool to room temperature and excess SOCI2 removed
invacuo. The residue was fractionally distilled bp 67-68' at 0.015 mmHg to give the title
compound as a colourless oil in 4.309 Q67o) yield.
79 (100). IR (thin
film):
MS:
165
(M-Cl*,0.5)'
135
(4)'94 (31),
lH
3300s (H-C=), 2928s,2852s,2140w (C=C), I796s (C=O).
NMR: I.32-L.55 (m, LOIH^, methylene protons); I.70 (quintet,2H',I 7 .zlfz, CHr-CF!-COC1);
1.93
(t,lH, .f 2.6H2, HC=); 2.tB (dt,2IJ, f 6.9,2.6IJ2, CH"-GC); 2'88 (t,2H,
CrL-COCI). "C NMR: 18.33,25.00,28.33,28.54,28.74,28.89,29.04,47.06
84.58 (alkynyl);
17
J 7 -2H2,
(alkyl); 68.14,
3.77 (carbonyl).
Undec-l0-ynamide (68)
Thionyl chloride (7.30g,4.5mI,61 mmol) was added dropwise to stired undec-lO-ynoic acid
(57) (9.33g, 51 mmol) at 40'. The mixture was refluxed for 60 minutes, cooled to room
temperature and added dropwise to concentrated NH, solution held at -15" (ice-MeOH bath).
The precipitated white product was collected, washed with water (20m1), air d¡ied and
recrystallised from CffrCL (charcoal). The product was recovered as colourless crystals mp
94-95" in7.84g (in two crops) (84Vo) yield. HRMS Calculated for C,,H,,NO: 181.1467.
Found: 181.1459. MS: 182 (M+H*, 2l),
(nujol): 3356
and 3184 s
L}l (M*, 2), 122 (21),72 (19),59 (100). IR
(N-H), 3280 s (H-C=), 2150 w (GC), 1662 and 1632 s (C{
amide). tH NMR: 1.31-1.65 (m,l2H, methylene protons); 1.93 (t,lH,.r 2.6H2,
H-e);
t'C NMR:
2.12-2.24 (m,4H, Ctq-C: and CHr-CONII,); 5.56 and 5.89 (2 x bs, 1H, CONIå).
20.36,27.48,30.41,30.64,30.79,31.00, 31.15, 37.92 (alkyl), 70.10, 86.71 (alkynyl), L77.83
(carbonyl).
Experimental 94
Experimental Described in Chaptet 2.2.
10- (Undec-
10-ynyl)-9-(10F)-acridone
(69)
: Method A.
1
2
3
4
To a stirred suspension of acridone (12) (1.00g, 5.1 mmol) in DMF (25m1) was added NaFI
(807o suspension in
oil,
169 mg, 5.6 mmol, 1.1
eq). The reaction mixture turned
a
fluorescenr yellow-green as stirring was continued at 50" for 60 minutes. The iodide (62)
(1.579,5.6 mmol, 1.1 eq) was added dropwise, and stirring continued at room temperature
for 60 minutes. TLC (40160 EtOAc/hexanes) of the dark green reaction mixture showed
spots for product
(& 0.55) and acridone (& 0.18); a fluorescent spot on the baseline was
assumed to be the acridone
salt. After 3 hours stirring the baseline spot was absent. The
reaction mixture was poured into water (100m1), the aqueous mixture extracted with CHCI,
(2x25 ml), organic extracts combined, washed with water (50m1), dried (NqSO), the
solvent removed and the residue recrystallised from EIOH to give the title compound as green
needle crystals in 0.409 (23Vo) yield mp 94-95". HRMS Calculated for CroFlrNO: 345.2095.
Found 345.2t01. MS: 345 (M*, 32), 208 (100), 195 (37),81 (35). IR (nujol): 3280s
(H-C=), 1638s (C=O), 7602s,1496s,1264s,1180s, 754s. UV (EIOH): 208 (46 500), 253
(66 300), 385 (S 900), 404 (9 800). Fluorescence (EIOH, À". = 385 nm): 421(100), 444 (75)
s/¿.
tH NMR: 1.29-1.52 (m, l2IJ, methylene protons); I.76 (quintet,2}I, J 7.5 H:z,
N-CIü-CH ,); 7.97 (r, lH, J 2.1Hz, C=C-H); 2.16 (dt,zH,
2}J, J
f
6.6, 2.3 IJz, CH"-GC); 4.1 1 (r'
7.8Ijr2, N-CIlr); 7.I7 (t,2IJ,J 7.5Hl2, C2-H); 7.33 (d,zH,18.4IJ2, C4-H); 7.60 (t,
t'C NMR: 18.1,26.5,26.7,28."1.,28.3,
zIJ,r 8.1H2, C3-H);8.48 (d, 2Ig,J7.9Hz,C1-H).
28.7
,28.9,29.1,45.7 (alkyl); 68.0, 84.3 (alkyne); 114.2,120.7,121.9,127.4,133.4, 141.2
(aromatic);
177
.4 (carbonyl).
Experimental
95
10-(Undec-10-ynyt)-9-(10l1)-acridone (69): Method B.
Acridone (12) (1.00g, 5.1 mmol), benzyltriethylammonium chloride (50mg,0.2 mmol), 507o
aqueous
KOH (10m1) and 2-butanone (10m1) were stirred at 60'fo¡ 30 minutes, then
1l-iodoundec-l-yne (62) (2.00g,7.1. mmol) was addeddropwise over 10 minutes. The
temperattlle was raised to 80" and the mixture stirred for 5 hours until analytical TLC
indicated the absence of starting material. The reaction mixture was poured into hot water
(100m1), allowed to cool to room temperature and then placed in an ice bath. The solid
which formed was collected and recrystallised from EIOH to give the title compound
green needle crystals mp95-97"
in 1.069 (59Vo) yield, with specral
as
light
data identical with those
reported in the previous procedure.
11-(1-Pyrenyl)-undec-10-yn'1-ol (70)
To a stired mixture of l-bromopyrene (1.00g, 3.6 mmol) in pyrrolidine (10m1) was added
undec-10-yn-1-o1 (59) (0.72g, 4.3mmol, 1.2eq), and Pd(PPhr)o (0.209, mmol, 0.05eq). The
reaction mixture was stirred at 80'for six hours, cooled to room temperature, poured into
saturated NH4CI solution (50m1), the mixture extracted with
CtlClr(2x
25ml), the combined
organic exrracrs washed with L07o citric acid (25m1), brine (25m1), dried (MgSO) and
solvent removed. The residue was separated by flash chromatography with 30n0
EtOAc/hexanes as the eluant and recrystallised from hexane to give the product as colourless
crystals mp 46-48" in 0.859 (65Vo) yield. HRMS Calculated for
Crr[rO:
368-2140. Found:
36g.ZtZ6. MS: 368 (M*, 100), 239 (50). 'H NMR: 1.48-I.52 (m,llFI, methylene and
hydroxyl protons); I.56 (quintet,2P',
f
6.5 IJz, fu-C=C-CfL-CHr-); 1.77 (quintet,2H, J 'l .2
Hz, -CH,-CF!-OH); 2.64.(t,zlir,17.0Il2, CH2-GC-At);3.62(t,2IJ,J 6.5Id2, CH,-OH);
7.97-8.20(m,ïH,Ar-H);8.56(d,LH,J9.}Hz,tu-H).
t'CNMR: 19.9,25.7,28.9,29.0,
Experimental 96
29.t,29.4,29.5,32.6,63.0 (alkyl);79.6,96.4 (alkyne); 118.8, 124.3,124.4,125.3,125.7
126.1,127 .2, L27
.7
,
127
,
.9,130.6, 131.2,131.8 (aromatic).
11-(1-Pyrenyl)-undec- l0-ynal (72)
Pyridinium chlorochromate (1.749, 8.00 mmol) and anhydrous NaOAc (0.669, 8.0 mmol)
werc suspended in CIrCl2(15m1) and a solution of the alcohol 70 (1.499,4.0 mmol) in
CÍJ2C12(10m1) was added
with stirring. Stirring was continued for 4 hours, at which time
analytical TLC of the da¡k brown mixture showed the absence of the alcohol. The reaction
mixture was filtered, the solvent removed and the residue purified by flash chromatography,
eluant z}tS}EtQAc/hexanes. The aldehyde was recovered as a viscous oil which slowly
solidified to a colourless solid mp 58-59" in 7.429 (96Vo) yield. HRMS Calculated for
crrg"uo: 366.1984. Found: 366.1989. MS: 366 (M*, 100), 253 (75),239 (48). IR (nujol):
3100w (Ar-H), 2750w (C-H aldehyde),224}w (C=C), I720s (C=O), 846s. 'H NMR:
1.36-1.69 (m, l2H,methylene protons); 1J7 (quintet,2}J, J 7.2H2, CHr-C[L-C=);2.42 (dt,
CI!-CHO);2.65 (t,2IJ, J 6.9 IJz, CH2-GC-Ar); 7.98-8 .21 (m,8H' Ar-H);
t'C NMR: 19.9, 22-1, 28-3, 28.9,
8.56 (d, lH, .,r 9.2Hz,Ar-H); 9.7 5 (t, llg, J 1.8 Hz, CHO).
2IJ, J 7.3, 1.8 Hz,
29.!,29.3,43.9 (alkyl),79.1,96.3 (alkynyl), 118.9, 124.4,124.4,124.5, L25.3,125.7 , L26.1,
127
1-
.2, I27 .7,
127
.9, I29.6, 1 30.6,
1
3 1.
1, 131.3,
1
3 1. 8
(aryl), 202.8 (C=O).
(1-Pyrenyl)-dodeca- 1'11-diyn e (7 4)
Triphenylphosphine (6.029,22.9 mmol) was dissolved in CH2CI, (20m1) and cooled to -15" in
an
iceMeOH bath. A solution of CBro (3.809, 11.5 mmol) in CF!C! (20m1) was added, the
cooling bath replaced by an ice bath and the mixture stirred at
0'for
30 minutes. A solution
of the aldehyde 72 (1.40g,3.8 mmol) in CHrCl, (10m1) was added, and the mixture stirred for
Experimental
t hour.
97
The solvent was removed and the residue purified by flash chromatography with
EtOAc/hexanes 5/95 as the eluant. The productT3, which was recovered in 1.969 (98Vo)
yield, was dissolved in THF (30m1), cooled to -78o in
a
dry ice/acetone bath and
n-butyllithium (3.0m1,2.5Min hexanes, 7.5mmo1) added dropwise via syringe. The red
mixture was stirred for 2 hours at -78o, t hour at room temperature and the reaction quenched
by the addition of saturated aqueous NH4CI (7ml). The mixture was separated, dried
(NqSO)
and the solvent removed. The residue was purified by flash chromatograPhy, eluant
EtOAc/hexanes 1/99, to give the title compound as an oil which slowly solidified to a
colourless solid mp 40-41" in 0.869 (64Vo) yield. HRMS Calculated for CrrHru: 362.2034.
Found: 362.2032. MS: 362 (M*, 15), 255 (38), 198 (62), 181 (100), 153 (58). IR (thin
film):
3300s
(H-G),
3040m
(tu-H), 2928s,2852s,2245w CGC-), 2140w (H-GC), 1600m,
1582w, !506w,1490w,1466m,1436m,1t86m,846s, 718s. UV (CHClt): 249 (47 000),264
(13 900), 274 (29 400),285 (54 900), 329 (17 200),345 (39 600),364 (56 000).
Fluorescence (CHCI'
L.*=
364
nm): 386 (100), 396 (63),406 (68). lH NMR: L.37-1.60
(m, L}H,methylene protons); I.76 (quintet,2H., J 7.2H2, CHrCÉl-=-Ar); 1.95 (t, IIJ, J 2.6
Hz, GC-H);2.L9 (dt,2IH,f 6.9,2.6H2, CIt-=); 2.64(t,2}ì,J7.2H2, CHr-:Ar);7'96-8'L9
(|n, 8H, Ar-H); 8.55 (d,
lH,,r 9.2H2, Ar-H). ttc NMR: 18.4, 19.9, 28.7,28-9,29.00,29.r
(alkyl),68.1,79.6,84.7,96.3 (alkyne);118.8, 124.3,724.4,125.7,126.1,127.2,I27.7,
127
1
.9, 129.6,130.6, 131.1, 131.2,131.8 (aromatic).
1-(1-Pyrenyl)-undecan-1-ol (75)
A mixture of 11-(1-pyrenyl)undec-10-yn-1-ol (70) (1.009, 2.7 mmol), 5VoPdlC (300mg) and
EtOAc (100m1) was stirred overnight under an atmosphere of hydrogen. The reaction
mixture was filtered through celite, the solvent removed and the residue recrystallised from
hexanes to give the product as colourless crystals mp
48'in 0.899 (897o) yield. HRMS
Experimental
98
CalculatedforÇrHrrO: 372.2453. Found 372.2465. MS: 372 (M*, L00),2L5 (24). IR
lH NMR:
(nujol mull): 34I6brs (O-H), 3050w (tu-H), 1610s, 1520s, 1054s, 836s.
1.25-1.52 (m,IfiIJ,methylene protons); 1.66 (ås,
(quintet,2IJ,
f
CH2-OH); 7.82
'l .5 IJrz,
lH
(exchanges
with
Dp), -OH); 1.81
CH2-CI{2-Ar);3.28 (t,2}J, J 7 .7 H:z, Ar-CHr); 3.57 (t,2IJ,
(d,lH,.r 7.8fI2, At-fÐ; 7.91-8.13 (m,'lH, At-ff);
8.23
f
6.6H:2,
(d,lH,,f 93rI2,
Ar-H). t'CNMR:25.7,29.4,29.5,29.7,29.8,3L.9,32.7,33.6,63.0(alkyl);123-5,124.5,
124.7,125.0,725.7,126.4,I27 .0, 127.2, 127.5, t28.5,129.6,130.8, 131.4,137.3 (aromatic)
11-(1-Pyrenyl)-undecanal (76)
The alcohol T5 was converted to the title compound by the method used for compound 72.
The title compound was isolated in 0.899 (897o) yield as colourless crystals mp 43'. HRMS
Calculated for
rH
CrrIloO: 370.2297. Found: 370.2290. MS: 370 (M*, 79), 215 (100).
NMR: 1.26-1.48 (m,l4H,
methylene protons); 1.83 (quíntet,2H, J 7.7 }Jz, CHr-CF!-Ar);
2.36(dt,2IJ,J7.3,L.9Ijrz,CH"-CHO);3.31 (t,2}J,J7-6H2, CI!-Ar);7.83-8.28 (m,9}f,
l',C NMR: 22.1,29.1,29.3,29.4,29.5,29.8,31.9,33.6
Ar-H); 9.72(t,1H,.I L.9Hz,CHO).
(alkyl), 123.5, L24.6,124.8,125.1,125.8,126.5,127 .L,127.2,127.5,128.6,129.7 ,130.9,
131.4, 137 .3 (aryl), 203.0 (carbonyl).
1-(l-Pyrenyl)-dodeca-ll-yne (78)
The aldehydeTí was converted to the title compound by the method used for compound 74.
The intermediate 1,1-dibromo-12-(l-pyrenyl)-dodec-l-ene (77) was isolated as colourless
tH
crystals mp 4L-43" in 0.879 (80Vo) yield. ll/rS: 524/526/528 (l:2:1,100, M*), 2I5 (2Ð.
NMR: L28-I.48 (m, l4H,
methylene protons); 1.87 (quintet,zIJ, J 7 .4H2, CHr-Ct!-Ar);
2.O8(q,zIH',J'l.OHz, CH2-CH=);3.35 (t,2IJ, CF!-Ar);6.38 (r, 1H,.I7.3Hl2, H-C=);
t'C NMR: 27.8,29.0,29.3,29.5,29.5,3r.9,33.0,33.6 (alkyl),
7.86-8.32 (m,9If{,., Ar-H).
Experímental
88.4
99
(BrrC-), 123.5,124.6,124.8,125.7,126.5,127.1,127 .2,127.5, L28.6,129.1,130.9,
131.5, I37.3 (aryl), 138.9 (HC=). The title compound was isolated in 430mg (717o) yield as
colourless crystals mp 50-51'. HRMS Calculated for CrrHro: 366.2348. Found 366.2334.
MS: 366 (M*, 49), 215 (100). IR (nujol): 3308s (H-G), 3040w (AI-H)' 1600s, 1180s,
842s
UV(EIOH):205(107000),234(143000),243(191 000),256(48300),266(96700),277
(160 000), 313 (40 C[r}),327 (86 700), 343 (117 000). Fluorescence @tOH, X".
= 343 nm):
377 (100), 397 (59),416 (20). 'H NMR: I.25-I.53 (m,l{H,methylene protons); 1.81
(quintet,2]fl^,J7.6Hz,tu-CfL-CHr);1.93 (r, lH, J2.7Hz,C=C-H);2.15(dt,2II,17.0,2.7
Idz, -CIJr-C=C); 3.31 (r, lH,
7H, Ar-H) ; 8.24 (d,
llfi,.,
r 7.8llJ2, Ar-CHr);
J 9.3 Hlz,
Af-H).
t3C
7.82
NMR:
(d,lH,,r
7.8 Hz, Ar-H); 7.92-8.13 (m,
18.4, 28.4, 28.7,
29.I, 29.5, 29.8, 31.9,
33.6 (alkyl); 68.1, 84.8 (alkyne); 123.4,124.6,124.7,I25.O,125.7,126.3,126.4,127.0,
!27 .2, 127 .5, 128.5, 129.6,130.9, 131.4, L37 .3,138.3 (aromatic).
Fluorescein methyl ester (80)
Fluorescein (5) (5.009, 15 mmol), concentrated FISO. (3ml) and MoOH (100m1) were
refluxed for 30 hours, and left at room temperature for 2 days. The orange crystals which
precipitated were collected, dissolved in 1.5N NaOH (50m1), extracted with EtOAc (50m1) to
remove the dimethyl derivatives, acidified to pH 1 with l}Vo}J:Cl, the red precipitate
collected and recrystallised from MeOH to give 1.329 (25Vo) of the title compound as red
microcrystals mp 282" (lit.t3o 282"). MS: 346 (M., 100),258 (46).
6-O-(1-Undec-10-ynyl)fluorescein methyl ester (81)
t0
4
,|
7
3
6'
5
A mixture of fluorescein methyl ester (80) (1.009,2.89 mmol), 11-iodoundec-1-yne (62)
(I.20g,4.33mmo1, 1.5 eq.), K2CO3 (0.809, 5.78 mmol,2eq.) and 2-butanone (100m1) was
refluxed for 8 hours. The mixture was filtered, the solvent removed and the residue dissolved
Experimental
100
in CHCI, (50m1). The organic solution was washed with 1.5N NaOH (50m1), water (50m1),
brine (50m1), ùied (NarSO.) and the solvent removed. The residue was recrystallised from
CflClhexanes to give 1.19g (797o) of the title compound
as bright orange crystals mp
83-84". HRMS Calculated for CrrHrpr: 496.2250. Found: 496.2234. MS: 496 (M.,97);
446(79);360(1@);25S(48). IR(nujolmull):3296s(H-G),3180w(tu-H),2145w(C={),
1730s (C=O),
l&4s,1590s, 1260s,852s. UV (EIOH): 234(135 000),256 (62700),277 (60
500),311(29400),364(29500),437 (74000),459(94400),489(80600). Fluorescence
tH
(ErOH, ?r", = 489 nm): 517 nm.
(quintet,zIJ,
f
NMR: I.34-1.56 (m, L2H, methylene protons);
1.83
6.6H2, CH2-CI{2-O); 1.94 (t, 1 2J Hz, H-G);2.19 (dt,2IJ,1 6.9,2.7
C[L-C{):3.64
(s, 3H, CHr-O); 4.06
}lz,
(t,2]l, J'1.0Ill2, Ct!-O-Ar); 6.a6 (d, J 1.9 }Jlz, C4-H);
6.54 (dd,.I 9.7, 1.9 IJz, C2-H);6.73 (dd,"f 8.6, 2.4H2, C7-g); 6.85 (d,
f
9.7 H:z, Cl-H); 6.88
(d, J 8.6 Hz, C8-H); 6.94 (d, J 2.4 Hz, C5-H);7 .31 (dd, J 7 .4, 1.4 Hz, C3'-H);7 .67 (dt, J 7 .4,
t'C NMR:
'1,.3H2,C5'-H); 7.74 (dt, J 7.4,1.3lf{r2, C4'-H); 8.25 (dd, J 7 -4,1.3Fir2, C6'-H).
18.4,25.9,28.4,28.7,28.9,29.0,29.2,29.9,52.4 (akyl), 68.1 (HC=C), 68.9,87 .7 (C=CH),
100.5, 105J,113.9, 11,4.6,Lr7.4,128.8, 129.6,129.8,130.2,130.3, 131.1, 132.7,134-7,
150.4, 154.3,159.0, 163.7 (alkene and aromatic), 165.7 (ester), 185.7 (carbonyl).
N- (l-Oxoundec- l0-ynyl) -S-aminofl uorescein (8a)
10
4
7
3',
I'
4'
6'
a
I
5
84
85
To a stirred solution of S-aminofluorescein (83) (0.50g,1,.44 mmol) in pyridine (5ml) was
added dropwise undec-lO-ynoyl chloride (67) (0.589, 2.88 mmol,2"q). The reaction mixture
was stirred for 48 hours, poured into water (50m1), acidified with l07o HCI until pH<2, the
bright orange precipitate collected, washed with water (5ml), air dried, dissolved in a small
quantity of EtOAc and precipitated by the additio
bright orange crystals mp 138-140'in 0.639 (657o) yield. Slow recrystallisation from
EtOAc/hexanes gave crystals composed of a mixture
of
lactone (colourless) and acid
(orange) forms. HRMS (LSIMS) Calculated for C'ItoNOu
(M+H.): 512.2073. Found:
5t2.7987. MS: 512 (M+H*, 100), 347 (16),207 (19). IR (nujol): 3500-2500m (O-H)'
3296s
(H-G),
2140w
(e{),
1738s, 1666s (C=O), 1610s. UV (EIOH): 231(48 500),255
(24900),455 (6 840),483 (6 670). Fluorescence @tOH, L". = 483 nm): 516 nm. The NMR
data are for the lactone structure
(8Ð. lH NMR (d6-DMSO):
1.30-1.44 (m,IDIJ, methylene
protons); 1.63 (quintet,2If,J 6.6H2, CHr-CII2-C(O)N); 2.14 (dt,2IJ,I 6.8,2.5}J2,
C[L-C=); 2.37 (t,2H, J 7 .3 IHz, CH2C(O)N); 2.73 (r, lH, J 2.5 IJz, H-GC); 6.54 (dd,zIJ,I
8.6,2.L}J2, C2-H);6.59 (d,2}j^, J 8.6H2, Cl-H); 6.66 (d,2}l,
f 2JHz,
C4-H); 7 .I9 (d'
J 8.3}j2,C5'-H); 7.82(dd,lH,,f 8.3, 1.8H2, C6'-H);8.33 (d,I}J,J I.$}jlz, C5'-H);
l}f'
10.11
(bs,2H,Ar-OH); 10.35 (bs, lH, C(O)NH). t'C NMR (d6-DMSO): 19.5' 26.8'29.7'29.9'
3O.2,30.4,30.5, 38.3 (alkyl), 72.8 (H;C=C), 84.8, 86.3 (HGC), 104.0, 111.6, L14.3,115.1,
126.1,128.0, 728.7,130.9, 742.6,148.4,t53.7,161.2 (aryl), 170.4, L73.7 (carbonyl).
5-Dimethylamino-N-(1l-undec-1-ynyl)-1-naphthalenesulphonamide (87)
6
54
3
2
7
8
1åu¡r------:
To a stirred mixture of dansyl chloride (S6) (200m9,0.74 mmol) andEgN (75mg, 100p1'
0.74 mmol, leq) in
1
"q).
CtlCl, (5ml) was added undec-10-yn-1-amine
(66) Q2amg,0.74 mmol,
The reaction mixture was stirred for 60 minutes, the solvent removed and the residue
separated by flash chromatoglaphy eluant 15:85 EtOAc/hexanes to give the title compound as
a fluorescenr green
oil in 215m g Q27o) yield. V/hen stored at -18" the oil solidified to give a
fluorescent grcen solid mp 58'. HRMS Calculated for CrHrrNrOrS: 400.2184. Found:
400.2200. MS (EI, Vorelative abundance): 400 (M*,77), 171 (100). IR
(thinfilm): 3300br
Experimental L02
s
(H-G
and
N-H),
3052w
(Ar-H), 2lI6w
(G{),
1586s, 1574s,1464s,1322s and 1144s
(O=S=O), 790s. UV (EIOH): 220 (29 900), 253 (12 400),337 (4 000). Fluorescence
(EIOH,7r,"*= 337 nm): 506nm.
lHNMR: l.O9-L.26(m,l2E,methyleneprotons);1.48
(quintet, J 7.5IFr2, CH2-CH2-NH);
CHr-e{X
l9a
Q' J 2.7 Hz'
H-C=);2.16 (dt,2H,J 7.0,2.7
2.88 (q,2iH,J 6.8HL2, CFL-¡fg); 2.89 (s, 6H (Cq)2-N); 4.59
SOrNH); 7.lg (d,IlH, J 7.7 IJz, C6-H); 7 .53 (dd,
8.6,7 .7 lFrz, CI -H);8.25 (dd,
lll,
lIJ,I
H4
(bt,lIJ,I5'2H2,
8-4,7.4 Hz, C3-H);7 '57 (dd,lIJ, J
J 7 .3, L-3 }Jz, C4-H); 8.29 (d, lH, ,f 8.6}12, C8-H); 8'54
(dd,L}d,18.4,1.0H2,C2-H). t'CNMR:18.3,26.3,28.4,28.6,28.8,29.2,29.4,43.3,45.4
(alkyl), 68.1,84.7 (alkynyl), 115.1, 118.6, 123.2,128.4,129.7 ,129.8,130.4,134.7,152.0
(aryl).
Experimental
103
Experimental Described in Chapter 2.3.
5-Bromo-1,10-phenanthroline (88).
65
4
7
3
8
910
1
2
Anhydrous l,l0-phenanthroline (6.209,34 mmol) was dried at 120" at 0.01mmHg for 3
hours, and cooled in a iceÂVIeOH bath. Fuming IITSO. (607o oleum) (30m1) was added
slowly, and the mixture allowed to come to room temperature with stirring. When the
phenanthroline had dissolved, Brr(2.75g,0.89m1, 17 mmol) was added and the mixture
heated at 120" in an oil bath for 19 hours. After cooling to room temperature the reaction
mixture was poured onto ice (250g) and neutralised with concentrated ammonium hydroxide
solution. The aqueous mixture was extracted with CHCL (2 x 100m1), the organic extracts
combined, dried (MgSO) and the solvent removed. The residue was separated by flash
chromatography on alumina, eluant CHClr/hexanes 50/50, and recrystallised from dry CHC13.
The title compound was recovered as colourless crystals mp 117-118" (lit7s. 118') in 4.629
(52Vo)
yietd. MIS 258/260 (M*, 100), 179 (M*-Br, 99),152 (40),125 (28). 'H NMR:
(dd, lE,.f 8.1, 4.3H2, C3-H); 7 .74 (dd,lH,.f 83,4.4H2, C8-H); 8.14 (s,
7.65
lH' C6-H); 8'18
(dd, l}J, J 8.2,1.8 Hz, CY-ff); 8.66 (dd, lIJ, J 8.3, 1.6 IJz, C4-H);9.20 (dd (panially
obscured.), l]f., J 4.4,1.8 Hz, C9-H);9.21 (dd (panially obscured), lH, J
trc NMR: 120.6,123.4,123.6,124.2,127
.7
4.3,l.6Hz, C2-H).
,728.6, 129.4, 134.9, 135.7,136.9,150.5,
150.7,
5-(1f -Hydroxyundec-l-ynyl)-1,10-phenanthroline (89)
N
A mixture of 5-bromo-1,l0-phenanthroline 88 (2.599, 10.0 mmol), undec-10-yn-1-o159
(2.O1g,12.0
mmol,l.2
eq.) and Pd(PPhr)o (0.589, 0.50 mmol, 0.05eq) was
stired at 70'in
Experimental
104
pyrrolidine (40m1) for 7 hours, at which time analytical TLC showed the absence of starting
material at t{ì- 0.80 (alumina plates, MeOIVCH2Cl2I0l90). The rÞaction mixture was poured
inro saturated NH.CI solution (100m1), extracted with
CI!C\(2x
100m1), the extracts
combined, washed with satumted NH4CI solution (3 x 50ml), brine (25m1), dried (MgSQ)
and solvent removed. The residue was purif,red by flash chromatography on alumina, using
CHCl/hexanes 50/50 as eluant. The product was recovered as a colourless oil which
solidified when stored at -18' to give colourless crystals mp 52-54" in2.3lg (66Vo) yield.
HRMS: calculated for CrI!.NrO: 346.2045. Found: 346.2032. MS: 346 (M., 8),
260
(13),232 (33),219 (62),194 (21), 83 (100). IR (thin film): 3600-3100s (o-H), 3050w
(Ar-H), 2220w (C=C). tH NMR: 1.34-1.72 (m,l4IJ, methylene protons); 2.58 (t,2}J,r
CFL-G); 3.63 (t,2H, CH2-OH);1 .60 (dd,llt,"f 8.1, 4-4H2, C3-H); 7.68 (dd,lH, 'f
8.3, 4.3}J2, C8-H); 7 .92 (s,lH, H6); 8.16 (dd,lH, 'f 8J, l.7Hz, C4-H); 8.72 (dd,1H, 'I 8'3,
t3C
l.|Hz,C7-H); g.l4 (dd,lH, 4.4,1.7lH2, C2-H);9.19 (dd,lH,,f 4.4,l.ïHz, C9-H)'
6.9ÙJ2,
"f
NMR:19.5,25.6,28.5,28.8,29.1,29.2,29.3,32.6,62.6(alkyl);77.0,96.8(alkyne);
120.58,
123.0,123.1,127.9,728.4, 129.8, 134.6, 135.3,145.6,145.7, t50.2,150.2 (aromatic).
5-(Dodec-11-ynyl)-1,10-phenanthroline (91): Method A.
To a stirred slurry of potassium ferf-butoxide (35mg,0.32mmo1, 1.1eq) inTFIF (10m1) at
-78'was added dropwise diethylmethyldiazophosponate 98 (56mg, 0.32 mmol, 1.1 eq). The
mixture was stired for 10 minutes then a solution of the aldehyde 95 (100mg,0.29 mmol) in
TTIF (5ml) was added dropwise. The reaction mixture was stirred overnight as the cooling
bath warmed to room temperature, quenched by the addition of saturated NaHCO3 solution
(2ml) and water (15m1) was added. The mixture was extracted with CfI"CLr(2 x 10ml), the
combined extracts washed with brine (20m1), dried (MgSO) an¿ the solvent removed. The
yellow residue was purified by flash chromatography on alumina with CHClr/hexanes 30:70
as eluant, and recrystallised
from hexanes to give the title compound as colourless crystals in
Experímental
47mg(487o) yield mp 73-74". Calculated for
CroFlr\:
344.2252. Found:
105
34-2245-M*
3M (M*,lm),217 (15),2o7 (19), 193 (40), 145 (51), 105 (83). IR (nujol mull): 3172s
lH NMR: 1.27-1.53 (m,l4H, methylene
(H-C=), 2130w (C=C), 1512m,872m,740s.
protons); 1.77 (quintet,2IJr,f 7.7Hz,CH2-Cft-At);1.92 (r, lH,
f 2.6Hz,H-C=);2..14(dt,
2lJ,f 6.9,2.6Hl2,CÍI'-G);3.08 (t,2H,J7.5Hl2, Cft-Ar);7-57 (s,lH' C6-H);7-58(dd
(obscured)
,
IIj,.,
J 8.4, 4.2H2, C8-H):7 .64 (dd, IH, J 8.4, 4.2H2, C3-H); 8.15 (dd, lH,
.,f
8.1,
(dd,lH, 'I 8.4, 1.6 IJz, C|-H); 9.11 (dd, l}t, J 4.3, l;7 IJz, C2-H);9.17
(dd,lIJ,J4.2,l.6Hz,C9-H). t'CNMR:18.4,28.7,29.r,29.4,29.5,29.5,29.6'29.7,30.1,
1.7 IJz, C4-H); 8.4t
32.6 (alkyl), 68.0, 84.8 (alkynyl), t22.7,123.1,124.8, t28.1,128.4 132.3,135.3,132.4,
I45.5,146.5, t49.5, L49.6 (aryl).
5-(Dodec-11-ynyl)-1,10-phenanthrotine (91): Method B.
To a stired mixture of the aldehyde 95 (0.58g, 1.7 mmol) and dimethyl-(1-diazo-2-oxopropyl)phosphonate (0.489, 2.5 mmol, 1.5 eq) in dry MeOH (7 ml) at 0o was added &CO,
(0.469, 0.34 mmol,2 eq). The mixture was stirred for
t
hour at 0'then at room temperature
ovemight. After quenching with saturated NH.CI solution (4ml), the organic solvent was
removed, Cll2cl2(25m1) and water (25m1) added, the organic layer separated, dried (MgSO),
solvent removed and the residue purified as in the previous procedure. The product was
tH NMR data identical with those reported in the
recovered in 0.349 (58Vo) yield with
previous procedure.
Büs
-1,10-Phenanthroline-5-[(dodec-1l-ynyl)-1,10-phenanthroline]rutheniumGl)
hexafluorophosphate (92)
5
4
3
3',
4'
2',
nlN
l0:
6'
7
8
2PF6
Experimental
106
A mixture of å¡s-1,10-phenanthrolineruthenium(Il)dichloride 90 (100mg,0.19 mmol, leq),
5-(dodec-l1-ynyl)-1,10-phenanthroline 91 (65mg, 0.19 mmol, leq), water (2ml) and MeOH
(lml) was stirred at 50" for 48 hours. The dark red-brown mixture
was filtered to rcmove a
black precipitate, the filEant concentrated under reduced pressure and a solution of
.
ammonium hexafluorophosphate (500mg) in water (2.5m1) added. The precipitated omnge
crystals were collected, air dried and purified by flash chromatography on alumina (the
compound decomposes on silica), eluant CHCI3 to give the title compound as a dark red glass
mp >150" in 140mg (687o) yield. Attempted recrystallisations from various solvents were
unsuccessful. HRMS Calculated for CorH*FuNrPt02Ru (M-PF..): 95I.2313. Found:
951.2321. MS (LSIMS): 951 (to'Ro ìvf*.PF;, 100), 806 (to'Ro MH*, 6l), 6251'02RuM* phen, 22). IR (nujol): 3300w (H-c=), 1640m,1040m,840s (P-F),722s. UV (CHCL): 257
tH NMR (do-DMSO):
(31g00), 423 (11600), 449 (12300). Fluorescence: 578 nm.
1.22-1.48 (n, methyleneprotons); 1.79 (quintet,2}J^,J'1.2H2,
CfL-üIr-At);2.1I (dt,2H,J
6.8,2.6H2, CI!-G);2.70 (t, lH, J2.6Hz,H-C=);3.24(m,2H,Clr-Ar);1.70-7-78(m,6ÍI,
C3-H, C3'-H, C8-H and C8'-H);7.99 (d, I}J,
f
53}J2, C4'-H); 8.06 (ln, 5H, C7'-H, C4-H and
C7-H); 8.19 (s, lH, C6'-H) ,8.37 (s, 4H, C5-H and C6-H); 8.66 (d, l}J, J 7 .2H2, C2'-H); 8.76
(d, 4I¡, f
8.21g,r2,
ttc NMR (do-DMSO):
Cz-H and C9-H); 8.84 (d, LrI, J 7 .4lgl2, C9'-H).
17.6,n.9,28.0,28.4,28.8, 28.8, 28.9,29.8,31.3 (alkyl), 70.8, 84.5 (alkynyl),125.2,126.L,
127.9,130.0, 130.1, 130.4,133.8, L34.7,t35.4,\36.6,136.9,140.0, 146.4,147.2,147.6,
151.5, 151.9, 152.3,153.0, 153.9.
5-(ll-Hydroxyundecyl)- 1'10-phenanthroline (93)
r-oH
A mixture of alkyne 89 (1.009, 2.89 mmol),l07o Pd/C (0.509),lÙVo HCI (5ml) and MeOH
(50m1) was stirred under a hydrogen atmosphere for 24 hours. The mixture was filtered
through celite, the organic solvent removed and the pH of the aqueous residue adjusted to >12
with lM NaOH. The aqueous solution was extracted with CHCL
(50m1), the organic extract
Experimental
107
washed with water (50m1), d¡ied (MgSOr, the solvent removed and the residue purified by
flash chromatography on alumina, eluting first with 50/50 hexanes/CHCl, to rcmove
impurities, then CHClr. The product was recrystallised from CClo to give the title compound
as colourless crystals
mp 117.5-119.0'in 0.799(787o) yield. HRMS Calculated for.
QrIloNrO: 350.2358. Found: 350.2362. MS:
350 (M*,53),349
(IM-F!C=OH.1, 60), 207 (94),193 (100). IR (thin
I620s,L564s,1424s.
film):
([M-H]*,39),3I9
3600-3100s (o-H), 3050w (Af-H),
tHNMR: l.2I-I.42(m,lfiH,methyleneprotons);
1.49 (quíntet,2H,J
6.9Hz,CH2-CH2-Ar); 1.70 (quintet'2H,J7.4IJ2, CH2-CIL-OH);2.O7 (brs' lH' R-OH);
2.99 (t,zlir,J 7.7
l, rz,
Cr!-Ar); 3.58 (r, 2H, J 6.6ljr2, CFIr-OH); 7.48 (s, lH, C6-H);7.50
IIJ,.I 8.0, 4.4 Hz, C3-H);7 .56 (dd, lIJ,
C4-H); 8.33 (dd, lH, ,f 8.4, 1.6
IJrz,
f
8.4, 4.3}J2, C8-H); 8.O7 (dd,
(dd,
lII, J 8'0, t'7 }lz,
Cl -H); 9.05 (dd, t}I, J 4.4, l jl IJz, C2-H); 9 'll (dd,
l}l,
r 43,1.6lH2, C9-H). t'C NMR: n.7,31.2,31.3,31.4,31-4,31.5,31.6,32-2,34-6,34-8,
64.9 (alkyl),124.6,124.9,126.8,130.0,130.4,134.2,137.3,139.4,147.5,148.5, 151.7, 151.8
(aromatic).
5-(11-Oxoundecyl)-1,10-Phenanthroline (95): Method A.
(cHtrocHo
To
a
vigorously stirred mixture of the alcohol 94 (573mg,1.64 mmol), NaBr (382mg, 1.64
mmol, 1.0 eq.), TEMPO (25mg,0.16 mmol,0.1 eq.), CITCI2(10m1) and water (2ml) at 0"
was added dropwise over 40 minutes a mixture of 0.35M NaOCI (4.7mI,1.64 mmol, 1.0 eq.)
and NaHCO, (382mg
,4.28 mmol, 3.0 eq.). The mixture was stirred fot 20 minutes, the
layers separated, the aqueous layer exracted with
combined, washed succesively with aqueous
CIlCl,
(10m1), the organic layers
KI solution (0.25gin 10ml),
107o NaSrO,
solution (10m1), brine (10m1) and dried (MgSOJ. fne solvent was removed and the residue
purified by flash chromatography on alumina using CHCI3 as eluant, to give the title
compound as crcam crystals mp 52-54" in2lTmg (387o) yield. HRMS (LSIMS) Calculated
for ÇIlnNrO (M+H.): 349.2280. Found: 349.2263. MS: 349 (M+H*, 72),321(53),207
Experimental
108
(82),194 (100). IR (thin film): 3030w (tu-H), 2716w (aldehyde C-H), 1724s (C=O), 1424s,
744s. tH NMR: l.2l-L.49 (m,lZId, methylene protons); l.6I (quintet,2H, r
7.l}lz,
CH2-CI{2-CHO); 1.79 (quintet,2H,f 7.2Hz, CH2-C[I2-Ar);2-41(dt,2H,J7-3,1.9IJ2,
CI{r-CHO); 3.11 (t,2H, J 7.5Hl2, CHr-Ar); 7.60 (s, lH, C6-H);7 .61 (dd, tIJ, J 8.1, 4.4H2,
C3-H); 7.67 (dd,lH',
f
8.4,4.3 Hz, C8-H); 8.18 (dd,
J 8.4,l.5lHz, C7-H); 9.13 (dd,
9.75
LPr,
f
t}l,J
8.1, l-4}J2, C4-H); 8.44 (dd,
l}J,
43,1.5 Hz, C9-H);9.19 (dd,l}J,J 4.4,1.4H2, C2-H);
(t,lH, J !.7 rfz,CHo). t'C NlvlR: 21.8,24.8,28.8,29.1,29.2,29.4,29.9,32.3,43.6
(alkyl), 122.5,122.9, L24.6,127.8, !28.2,132.0,135.1,
I37
.2,I45.2, 146.2,149.2,149.3
(aryl), 202.7 (carbonyl).
5-(11-Oxoundecyl)-1,10-Phenanthroline (95): Method B.
Oxalyl chloride (0.22m1,2.6 mmol. 1.1 eq) was dissolved in
Ct!C!
(25m1) and cooled
in
a
dry icelacetone bath. DMSO (0.36m1, 5.1 mmol, 2.2 eq) was added and the reaction mixture
was stired for 5 minutes. A solution of the alcohol 94 (815mg, 2.3 mmol) in
CIICI,
(10m1)
was added, the mixture stir:red for 15 minutes, EgN (1.62m1, 11.6 mmol, 5 eq) added and
stirring continued. for 5 minutes. The cooling bath was removed and the reaction mixture
allowed to come to room temperature. W'ater (25m1) was added, the organic layer was
separated, washed
with brine (25m1), dried (MgSOJ an¿ solvent removed. The residue was
separated by flash chromatography on alumina, eluant CHC13 to give the title compound
in
559mg (69Vo) yield. The physical data were identical with those in the previous method.
Diethylmethyldiazophosphonate (98)
(
a) Phthalimidodiethylphosphonomethane
4
5
6
'1
3
{Hz-
o
ll
Pi-oFr
-OFJ
1
To a stirred solution of bromomethylphthalimide (6.009,25 mmol) in xylene (mixture of
isomers,20ml) at 130" was added dropwise triethylphosphite (4.159,25 mmol). The
byproduct ethyl bromide was distilled from the reaction mixture over 60 minutes, the
Experímental
109
temperatue raised to 150' and stirring continued overnight. After cooling to room
temperature hexane (20m1) was added and the reaction mixture stored at -20". The
precipitated crude product was collected, washed with hexane, air dried and recrystallised
from ether. The title compound was recovered as colourless crystals mp 63-65" (litttt.
tH
64-65") in 3.839 (in two crops, 48Vo) yield.
(d,2njrnl!.5Hz,P-Cfl"-$;
NMR: 1.32 (t,6}I, J 7.l}lz, CHr-CF!); 4.10
a.20 (m,4H,o-cH2-cH3);7.73 (d"d'2H'15.6'3.0IJ2' C5-H
and C6-H);7.87 (dd,2]H,J 5.6,3.0lfir2,C4-H and C5-H).
33.2(I*t56Hz,p-CII2-N),
62.8
t'C NMR: 16.3
ff* e.: Hz, CI[),
(Ie5.4Hz,O-CrL-CH3),123.5,131.9, 134.2 (aromatic),
166.9 (carbonyl).
(b) Diethylrnethyldiazophosphonate (98)
A mixture of phthalimidodiethylphosphonomethane (2.59,8.4 mmol), hydrazine hydrate
(0.429,8.4 mmol), acetic acid (1.01g, 16.8 mmol) and MeOH (10m1) was refluxedlot 2
hours. After cooling to 0o, the precipitated phthalhydrazide was filtered off and the solvent
removed. The residue was dissolved in
a
mixture of acetic acid (1.0m1) and water (8.5m1).
Dichloromethane (6mt) was added, the mixture cooled to -10' (ice-MeOH bath) and a
solurion of NaNO, (0.58g, 8.4 mmol) in water (1.5m1) added dropwise. The cooling bath was
replaced by an ice bath and the reaction mixture stired for 90 minutes. The organic layer
was separated, the aquoous layer extracted with CH2CL (3 x 10ml), the combined extracts
washed wirh saturated NaHCO, solution (20m1), brine (20m1), dried (tvtgSOJ and solvent
removed. The residue was distilled behind a safety shield, bp 64'at 0.012mmHg (Kugelrohr;
air temperature; lit.r3r 51o at 0.lmmHg) to give the title compound as a yellow oil in 0.949
(637o) yield. IR (thin
6IJ,
film):
r 7 J Hz,CHr-Ct!);
lH
2984s,2712s (Nr), 1300s and 1250s (P=O).
NMR: t.34
3.77 (d, !H,2Jw 1 1.0 Hz, HC=Nz)i 4.L3 (m,4H, O-CH2-CH3).
5-(11-Oxoundec-1-ynyl)-1'l0-phenanthroline (104)
(t,
Experimental
110
The titte compound was preparcd by Method B used for compound 95. Reaction of alcohol
91 (1.509,4.33 mmol) gave the product as colourless crystals mp
50-5f in 1.119 (69Vo)
yield. HRMS CalculatedforÇHrNp: 3M.1889. Found: 344.1886. MS: 344(M*,8),
315 (7), 259 (52),23I (69),219 (70),91 (100). IR (nujol): 2720w (C-H aldehyde),2212w
(C=C), 1722s (C=O), 1504m, 1420m,7$m.
protons); 2.38 (dt,
(dd,IIJ,J
2IH^,
tH NMR: 1.33-1.73 (m,l2H, methylene
J 7 .3, 1.7 IJrz, CIL"-CHO); 2.55 (t, 2IJ, J 7 .0 Hlz, CIL-C=C-Ar) ; 7 .56
8.1, 4.3II2, C3-H); 7.65
(dd,lH,,f 8.2H2,4.3II2, C8-H); 7.88 (s, lH' C6-H);
8.L2(dd,lIFr,J 8.1, 1.7H2,C4-H);8.67 (dd,I}J,I8-2,1-7}Jz,C7-gX9.ll(dd'lH,J 4'3,
t'CNMR:
1.7 }Jz,C2-H);g.16(dd,lH,,r 43,1.7 Hz, C9-H);9.72(r, 1H, f 7.6Hz,CHO).
19.2,21.3,28.2,28.3,28.4,28.5,28.6,28.8, a3.a (akyl),67.7,96.4 (alkynyl), 120.3, 122.8,
122.9,128.8, L29.2,129.6,134.4,135.1, t45.4,145.5,150.0, 150.1 (aryl),202.3 (carbonyl).
5-(Dodec-1,11-diynyl)-1'10-phenanthroline (105)
Prepared by the method used for compound 95 from aldehyde 104 to give the title compound
as colourless crystals mp
86-88'in 300mg (60Vo) yield. HRMS: Calculated for CroHrNr:
340.1939. Found: 340.1937. MS: 340 (M*, 88), 245 (38),219 (87)'231 (48),217 (75),190
(24),l4g (19),41 (100). IR (thin film): 33Nm (H-G), 302tw (Ar-H), 2928s,2852s,2220w
THNMR: 1.26-L.68 (m,10Il,
CC=C-), 2l2tw (H-GC), 1606m,!590m,1506s, 1424s,742s.
methylene protons); I.75 (quintet,zIJ, J 7 .2H2, CHr-CF!-=-Ar); 1.95 (r, lH,
H-G);
2.2O (dt, 2IJ,
f
6.9, 2.6 Hz,
CF!-È{H);
2.61 (t 2IJ, I
7
.0 IJz,
I 2.6}12,
CIt-=-Ar) ; 7 .63 (dd,
lH,,f 8.L,4.4 Hz, C3-H);7.71(dd,IIl,.f 8.3, 4.4H2, C8-H); 7.95 (s, lH, C6-H); 8.\9
f
(dd,
(dd,lIJ,J 8.3,1.8 Hz, CY-H); 9.17 (dd,lH,,r 4.4,l-8}l2,
t'CNMR: 18.4,19.7,28.4,28.7,28.7,28.9,29.0
c2-H); g.2O(dd,lH,J 4.4,1.8H2,C9-H).
lIJ,
8.3, 1.8 Hz, C4-H);8.74
(alkyl), 68.1,77 .2,84.7,96.9 (alkynyl), 120.2,123.2,123.3,128-I,128.6,130.0,
1.35.5, 145.8, 145.9, 150.4, 150.5 (aryl).
134.8,
Experimental
111
Experimental Described in Chapter 2.4.
Biotin N-hydroxysuccinimide ester (106)
3
I
4
I
5
H
Biotin 4l (0.979,4.0 mmol) was dissolved in DMF (12m1) at 80" and the solution removed
from heating. NHS (0.47g,4.1 mmol) and DCC (0.939, 4.5 mmol, 1.1 eq) were added and
the reaction mixture stired at room temperature for 2 hours, filtered to removed precipitated
DCU and the solvent removed under vacuum (oil pump). The residue was recrystallised from
isopropanol to give the title compound as cream crystals mp207-209" (lit.88 210") in 0.719
(Stvo) yield. MS: 341 (M*, 31), 227 (32),166 (11),97 (49),55 (100). IR (nujol): 3232m
lH NMR (d6-DMSO): 1.41-1.68 (2,
(N-H), 1750s, 1730s, t704s (3 x C=O), L2l6s,1072s.
6H, methylene protons);2.56 (d, l}J,
Js
I2.4 Hz, CS-Hb);2.65
(t,2IJ,I7
-3IJ2, CI!-CO,);
2.8I (dd, f s 12.4,4.6Pr2, C5-Ha); 3.10 (dt,l}J, J 7.3,4.6}J2, C2-}l);4.13 (dd,lH, J 7.0,
4.7 ldz, C4-H); 4.29 (dd,lH, J 7.0,4.7 Hz, C3-H);6.36 (ås, lH, Nl'-H); 6.42 (bs, lFI,
N3'-H). t'C NMR (d6-DMSO): 24.2'25.3'27.4'27.7 '29.8,39.8, 55.0, 59.0, 60.8 (alkyl),
162.5, 168.8,
B
17
0.2 (carbonyl).
iotin-N-(undec-l0-ynyl)ami de (107)
3
1
H
t!-(cH)r
H
3
5
To a stirred solution of biotin-NHs ester (106) (300mg,0.29 mmol) in DMF (6ml) was added
the amine 66 (147mg,0.29 mmol, 1eq). A white precipitate formed within 10 minutes. The
mixture was stirred overnight, the solvent removed invacuo (oil pump), the residue purified
by flash chromatography using SlgZMleOHlCII"Cl"as eluant, rccrystallised from MeOIIÆIO
and dried under vacuum to give the title compound as a colourless solid mp 176-178" in
Experimental
1,12
3l2mg(9o7o)yield. HRMS: CalculatedforÇ,[rNrOrS: 393.2450. Found: 393-2458.
MS: 393 (M*, 3), 333 (28),160 (100). IR (nujol): 3700-3200n (N-H), 3292s (H-C=),
tH NMR: 1.18-1.67 (m, ZÛH,methylene
21.4Ow(GC), 1692s, 1648s (C=O), 1548s.
protons); 1.87 (r, LH, J 2.6HL2, H-C=); 2.08-2.15 (m,4IJ, CIL-CON and
lH,,rs* l2.9Hz,CS-Hb); 2.86(dd,lH,,rs*
Crt-G);
?.66 (d,
12.9,4.9 Hz, C5-Ha);3.10 (dt,t}J,J7.1,4.6
}Jz,C2-H);3.L6(q,2lFr,J6.9Hz,CH2-NHCO):4.26(dd,IH,I7.7,4.9Hz,Ca-p;4'45(dd,
lH,J7|llfrz,4.6Hz,C3-H);4.56(bs,1H,Nl'-H);5.12(bs, lH,N3'-H);5.45(bt,lH,J5'2
trc NMR: 16.5,24.0,25.1,26.5,26.6,26.7,26.8,27.2,27.4,27.6,27.8,34.0,
Hz, -NHCO).
37
.3, 38.6, 54. 1, 5 8.0, 59. 8 (alkyl),
67
.9,
1
0
1.0 (alkynyl),
1
6
1.6, t7 0.9 (carbonyl).
Experimental
113
Experimental Described in Chapter 3.LProtection of Amino Acids: General Procedure
The crude amino acid was suspended in dry MeOH (10mVg) and SOCI (leq) added dropwise
with stirring. After stirring f.or 24hours, the solvent was removedinvacuo, and the crude
amino acid methyl ester hydrochloride suspended in a mixture of
CtlCl,
(25mVg) and
benzoyl chloride (leq). A solution of NarCO, (leq) in water (SmVg) was added dropwise and
the reaction mixture
stired for 48 hours. The organic layer was separated, washed with
saturated NaHCO3 solution, brine, dried
(MgSO), the solvent removed and the residue
purified by recrystallisation or chromatography.
N-Benzoyl-3-iodo-L-tyrosine methyl ester (108)
The crude product was tecrystallised from EtOAc/hexanes to give the útle compound as a
colourless glass in 0.569 (597o) yield. Calculated for C,rH,uINOo: 425.0L24. Found:
425.0t35. MS: 425 (M*,0.1),305 (M.-PhCONH, 100),233 (58). IR (nujol): 3500-3000n
tHNMR: 3.06and3.14(2xdd,lIJ,
(O-H), 1732s(C=Oester), 1640s(C=Oamide), l532s.
J l4.g,5.4Hz,Cf!-Ar);
3.71 (s, 3H, CO2CI!); 4.96 (dt,LIJ,.17.4,5.4IJz,, crC-H); 5.4I (bs,
lH, AI-OH); 6.54 (d, lH, J 7 .4Hz,NH); 6.81-7 -69 (m,8H, Ar-H).
t3C
NMR: 36-6,52'6,
53.6 (alkyl); 85.a (Ar-I), 115.1, 127.1,128.7,129.7,130.9, L32.0,I33.6,I39.1,154.4
(aromatic);
167
.t,
17
1.9 (carbonyl).
N-Benzoyt-4-iodo-r.-phenylalanine methyl ester (109)
The crude compound was recrystallised from CFlC{hexanes to give the title compound
as
colourless crystals mp 147-148" ln}.22g(73Vo) yield. Calculated for C,rHtuINOr: 409.0175.
Found409.0160. MS: 409 (M*,7),349 (M.-COrMe, 10),289 (M.-PhCONH, 34),287
rH
(100), 257 (31),217 (21). IR (nujol): 3308m (N-H), 1746s and 1640s (C=O), 1528s.
NMR: 3.18 and 3.26 (2 x dd, !H, J L3.9,5.6Hl2, Crl-Ar); 3.78 (s, 3H, CtL-OrC); 5.08 (dr,
lH, J 5.6,7.3 Hz, ctC-H); 6.15 (d,lIJ,J 7.3}ll2, PhCONH); 6.88 (d,2H,I 8.2H2, C3-H,
c5-H); 7.42-7.55 (m,3H, PhCON); 7.61 (d,zV, J 8.2H2, C2-H, C6-H); 7.12-7.75 (m,2H,
Experimental lL4
phCON). ',C NMR: 37.3,52.5,53.3 (alkyl);92.7 (Ar-I), L26.9,t28.3,128.6,130.0, 131.3'
131.6, 131.9, 133.3,133.6,135.5, 137.6 (aromatic); 166.8, L7I.7 (carbonyl).
N-Benzoyl-r.-tyrosine methyl ester (110)
The crude compound was recrystallised from EtOAc/hexanes to give the title compound as
colourless crysrals mp 149-152" (1it.t32151-153') inl2.9g(55Vo) yield.
Ms:
299 (M*, 13),
282(20),240(33),178 (63), 122(29),105 (100). IR(nujol): 3340bs (O-H) 3284s (N-H)'
THNMR: 3.l2and3.22Qx
3070w,3050w, 17l2s(C=Oester), 1640(C=Oamide), 1598s.
dd, 1H,13.8, 5.6
lFrz,
CHr-Ar);3.77 (s, 3H, CO'CE); 5.06 (dt,1H, "I 7 '7, 5'6 Hz, aC-H);
6.20 (bs,lH, AI-OH); 6.66 (bd,IIjr, J 7.7 IJrz, CONH-); 6.70-6.77 (m,2IJ, C3-H and C5-H);
6.94-7 .01
(m,2H, C2-Hand C6-H);7.37-
7
J5 (m,5H, PhCON). "C NMR (d.-DMSO):
37.3,53.6,56.5(alkyl), 116.8, 129.2,129.5,130.0, 131.8, 133.2,135.5, t57.7 (aryl), 168.2,
174.I (carbonyl).
N-Benzoyl-5-hydroxy-L-tryptophan methyl ester (111)
The crude compound was purified by flash chromatography eluant EtOAc/hexanes 50/50 to
give the title compound as a colourless glass in0.77g(50Vo) yield. HRMS Calculated for
c,#,,Nroo:
338.1267. Found: 338.1267. MS: 337 (M*, 32),
n8 6),216
(58), 145 (100).
tH NMR:
IR (nujol): 3600-3200n (Ar-OH str), 1732s (C=O ester), 1642s (C=O amide).
3.30 and 3.38 (2 x dd, lIH^,I lI.7 , 5.3ljr2,
CI!-Ar);
3.70 (s, 3H, CH3O2C); 5.1
|
(dt, J 7.7 , 5.3
Hz, aC-H); 5.86 (ås, lH, Ar-OH); 6.75 (d, LH' J 7.6IJ2, C(O)NH); 6.78 (dd,lH, .I 8.7,2.4
Hz, C6-H) ; 6.97 (m,2H, C2-H and C4-H);7 .2\ (d, l}J, J 8.7 }Jz, O-ff)t 7 '36 (m,3H and
t'C NMR: 27 -8,52.5,53.6 (alkyl), 103.0,
7.69, m,2H, PhCON); 8.14 (bs, lH, Nl-H).
109.3, t!2.0,112.3,123.8, !27.!,128.3,128.6,137.2,131.8, 133.6,150.2 (aryl), 167.3,
172.6 (carbonyl).
N-Benzoyl-(4-O-triftuoromethanesulphonyl)-L-phenylalanine methyl ester (112)
To
a
cold (0") stirred solution of N-benzoyltyrosine methyl ester (110) (1.00g, 3.7 mmol) and
EqN (0.34g,O.47m1,3.3 mmol) in dry CryCl2(15m1) was added N-phenyltriflimide (I.43g,
Experimental
115
4.0 mmol). The reaction mixture was stired overnight as the ice bath melted and came to
room temperature. The solvent was removed and the residue separated by flash
chromatography with 30:70 EtoAc/hexanes as eluant to give the title compound as colourless
crystals mp 114-115' (lit.8e 112') in 1.44e Q97o) yield. MS: 431 (M*, 0.1), 372 (M.-COrMe,
7), 310 (M.-CESOr, 100), 225 (55),192 (14),177 (35). IR (nujol): 3320m (N-H str), 3050w
lH NMR: 3.26 and3.34 (2x dd,
(Ar-H str), 1736s (C=O ester), 1638s (C=O amide), 1534s.
lH, ,f 13.9, 5.6 IFrz, CH"-Ãr);3.77 (s, 3H, COTCHT); 5.09 (dt, I}J, f 7 '2,1 5'6}J2, crC-H);
r'CNMR: 37.3,52.6,53.4(alkyl),
6.65(d,lH,.f 7.2H2,N-fÐ;7.L7-7.75(m,9H,Ar-H).
115.5,
l2l.4,lzt.g
and 128.6 (central peaks of 4,
132.0, 133.6, 1 36.8, 148.6 (aryl), 166.9,
17
CF' IcF334Hz),126.9,128.7,131.1,
1.7 (carbonyl).
N-Benzoyl-5-O-trifluoromethanesulphonyl'L-tryptophan methyl ester (113)
H
I
7
2
3
H
CO2Me
6
,
+
OTT
The title compound was prepared as described for compound 112. The crude product was
separated by flash chromatognphy eluant 40160 EtOAc/hexanes and recrystallised from
C1"Cl"lhexanes to give the title compound as colourless needle crystals in 0.639 (9IVo) yield
mp 109-110'. CalculatedforÇË,JrNrOuS: 470.0759. Found: 470.07M. MS: 470(M*,
g),4I1 (M*-CO2Me' 5)' 349 (M.-PhCONH2' 100)'278 (90)'225 (60). IR (nujol):
3403s
tH NMR: 3.38 and
(Ar-N-H), 3350n(N-H), 3050w, 1734s (C=O ester), 1636s (C=O amide).
3.46 (2 x dd, lIH,
J
14J , 5.3IFr2, CFI-Ar); 3.73 (s,3H, CI{3O2C); 5.13 (dt' I}J'.1
7
.3' 5.3H2'
aC-H); 6.78 (d,lH, J 7 .3lHt2, PhCONH);7 .03 (dd, J 8.8,2-4 Hz, C6-H); 7'08 (d, l}J, J 2'4
1¡z,C-4H);7.26-732
(m,71ti^,
At-IÐ; 8.68 (bs, lH, Nl-H). "C NMR: 27.5,52.7,53.3
(alkyl), 110.8, 111.1, 112.5,I75.4,123.3and127.1,(centralpeaksof q,CFp Jo283IJz),
125.6, L27.0, 127 .9, 128.7, 129.5,I32.O,133.5, L43.5 (aromatic), 167.2,172.3 (carbonyl).
Experímental
116
methyl ester
N-Benzoyt-4-{t11-(10-(10H)-9-acridonyl)lundec-1-ynyl}-L-phenylalanine
(118): Method A.
32
2'
corMJ
H
1
3'
s'
To a stired solution of triflate 112 (50mg, 0.16 mmol) in DMF (1.5m1) was added Pd(PPq)4
(134mg, 0.16 mmol). A green/brown suspension slowly formed. TLC analysis after 3.5
hours showed a minor amount of starting material at Rf = 0.32 Q0n0 EtOAc/hexanes) and a
major spot on baseline. Alkyne 69 (60mg, 0.l7mmol, 1.5eq), CuI (4.4 mg, 0.023mmo1,
0.2eq) and EgN (0.5m1) were added and stirring continued at
50'for 30 minutes. The solvent
was removedínvacuo and the residue separated by flash chromatography eluant 50/50
EtOAc/hexanes, to give the title compound as light green crystals in 5lmg (68Vo) yield mp
66-67". HRMS Calculated for Co,HorNrOo: 626.3145. Found: 626.3148.
MS:
626
(M.,5),
208 (9), 195 (100), 167 (25). IR (nujol): 3272w (N-H), 1732s (C=O ester), I642s (C=O
amide), 1606s, 1538s. UV (EIOH): 257 (87 700), 387 (10 400),405 (12 400). Fluorescence
(EIOH,
\"*=387 nm): 420 (100), 440 (70). tH NMR: I.l7-1.65 (m,l2H, methylene
protons); 1.92 (quíntet,
3.19 and 3.27 (2x
2I1r,
f
7
.6 Hz, CHr-CFl-acridone); 2.40 (t, 2H, .I 7 .0 }Jz, CHr-=-Ar);
dd,lIJ, ,f 13.8, 5.6ljr2, CHr-Ar); 3.75 (s, 3H, CH3O2C); 4'31 (m,2H,
CH,-N); 5.07 (dt,lH,J 7.4,5.6H2, aC-H);6.67 (d'l}J,J7-4Hz,N-ff);7.04(d,2H,J8'2
IJz, C3'-Hand C5'-H);7.25-7.33 (m,4IJ, C2-H, Cz'-H and C6'-H);7 .38-7.52 (m,5H, C4-H
and
PhcoN); 7.69-7.75 (m,4H, C3-H
and
PhCoN); 8.57 (dd,2H,,r 8.0, l.6Hz, Cl-H).
NMR: 19.3,26.8,27 .1,28.6,28.8,29.0,29.2,29.4,37 .6,46.1,52.4,53.4 (alkyl),
t3C
80.2, 90.6
(alkynyl), 114.5,72!.1,122.4,122.8,126.9,I27.9,128.6,129.2,131.6,131.8, 133.7,133.8,
135.3,141,.7 (aryl), 166.7, L71.8, L77.9 (carbonyl).
N-Benzoyt-4-{tl1-(10-(10If)-9-acridonyl)lundec-l-ynyl}-L-phenylalanine
(118): Method B.
methyl ester
Experimental
ll7
To a stirred mixture of DMF (1.0 ml) and EgN (0.2 ml) was added sequentia[y the iodide
109 (50mg,O.lzmmol), alkyne 69 (63mg,0.18 mmol, 1.5 eq), Pd(PPh3)4 (14mg'
0.012mmo1, 0.1eq), and CuI (4.5mg, 0.024mmo1, 0.2eq). After stiring overnight at room
temperarure TLC @tOAc/hexanes 50/50) showed the absence of of 109 at Rf 0.65 and new
compounds at Rf 0.51 and Rf 0.37. The mixture was separated by flash chromatography
using ETOAC/hexanes 40160 as eluant. First to elute was the alþne dimer 114 in 2.0mg
yield. [lH NMR (300 MHz,
õ
ppm): 1.23-1.54 (m,6IJ, methylene protons); 1.95 (quintet,
ly¡,.I7.8Hz,CHrCI{r-N);2.24(r, lH, J6.7Hz,CH2-=);4.33(m' lH'CFI2-N);1.29(t,2H,J
7.lHz,c2-H); 7.49 (d,zlfl^, J 8.7 IJz, C4-H); 7.74 (ddd,zH, J 8.7,7.1,I.7
}Jz, C3-H); 8.59
(dd,2H,,f 8.1, 1.7 Hz,Cl-H.)l Next to elute was the title compound, which after
lH NMR data were
recrystallisarion from CllClrlhexanes in 74mg(96Vo) yield. The
identical with those reported in the previous method.
2
1
4
tl4
Compounds 119 (Method A), 120,
l2l, \22,
123, 124 and 125 were prepared in a similar
manner, except for the difference stated.
Attempted formation of 118 by reaction of triflate tL2 with alkyne 69 using
PÇdbar/AsPh, catalyst.
A mixture of PÇdbq (24mg,0.026 mmol), AsPh, (52mg,0.I7 6 mmol) and DMF (3ml) was
stirred at room temperature until a yellow-brown solution was formed, then triflate
ll2
(100mg, 0.23 mmol), alkyne 69 (200mg, 0.579mmol), CuI (18mg, 0.095mmol) and EqN
(100p1, 0.69mmol) were added and the reaction mixture stirred at room temperature for 60
minutes. TLC analysis showed the absence of product, so the temperature was increased to
Experimental
118
50" and stiring continued overnight. TLC analysis then showed the absence of triflate, no
spot for coupled product 118 and a large spot coresponding to the coupled alkyne 114.
Attempted formation of 118 by reaction of triflateLl2with alkyne 69 using piperidine.
A mixture of triflate 112 (100mg,0.23mmo1), aþne 69 (96 mg,0.28 mmol, l.2eq),
pd(pph3)4 (20mg' 0.017 mmol)'
cul (20mg,0.11 mmol), PPL (20 mg, 0.08 mmol) and
piperidine (aml) was refluxed for 4 hours, at which time TLC showed the absence of triflate.
The reaction mixture was diluted with
CIlCl,
(25m1), the organic layer washed
with l07o
HCI (2 x 20ml), warer (20m1), dried (Na"SO) an¿ solvent removed. TLC analysis showed
the presence
of starting atþne 69 and alkyne dimer 114, and the absence of the coupled
product 118.
N-benzoyl-5-{11-t10-(10ä)-9-acridonyllundec-1-ynyl}tryptophan methyl ester (119):
Method A
H
I
7'
6'
4'
I
4
32
Reaction of triflate Ll3 (23mg, 49 pmol) with alkyne 69 (32mg, 92pmol) under the
conditions described for compound 118 (Method B) at 70' overnight, removal of solvents in
vactto, and separation by flash chromatography, gradient eluant 40160 to 60140
EtOAc/hexanes gave the title compound in 7.3mg (2l%o) yield. MS: 689 (lvl+H., 35),453
(100). tH NMR: 1.40-1.62 (m, L2H, methylene protons); 1.92 (quintet,2H,J 7 -6H2,
cH2cII2-N);2.39
(t'2H'r
6.9IJ2, CrL-=); 3.38
(2xdd,lIJ,
.,r
15.0, 5.2Hz,CHr-Ar);3.72
(s,3H, CI!O,C); 4.30(m,2H, C[L-N);5.t2(dt,l}J,J7.6,5.2Hz,crC-H); 6.70(bd,lH,.r
7.6Hz,PhCONH);6.97 (d, lH, I2.3Hz, C4'-H);7.18(d,zH''JO.9Hz,Ar-H);7.27 (t,2H,J
7.5lJ2,Cz-}l|);7.35-7.47 (m,4}l, Ar-H);7.48(d,2}J,J 8.1H2, C4-H);7.62(s,lH' C4-H);
Experimental ILg
7
.69-7 .75 (m, 4]H,
tu-g);
8.50 (bs,
lH, N1'-H);8,57 (dd,zlH,-f 8.1, 1.8 Hz, C1-H). t'C NMR:
19.4,26.8,27.1,27.5,28.8,28.8,289,29.2,29.3,46.2,52-4,53.4
(alkynyl), 109.9, 111.3, 114.6,115.1, 121.2, 122.3, 122.4,
123.7
,
(alkyl)' 81.5,87.5
125.7
, I27 .1,
127
.4,
L27 .9,
128.5,131.6, 133.8, 133.9,135.3,141.7 (aryl), 167.0,172.2,179.5 (carbonyl).
N-benzoyl-5-{11-t10 -(L0í)-9-acridonyllundec-1-ynyt}tryptophan
methyl ester (119):
Method B
A mixture of triflate 113 (100mg,O.2lmmol), alkyne 69 (110mg,0.32 mmol,
Pd(PPh3)4 (25mg,0.021
1.5 eq),
mmol,0.1 eq), CuI (8.lmg,O.O42 mmol,0.2 eq), PPh, (1lmg,0.042
mmol, 0.2 eq) and piperidine (5mI) was refluxed for 5 hours, at which time TLC showed the
absence of the
triflate. The solvent was removed in vacuo
and the residue subjected to flash
chromatography using eluant 40l60EtOAc/hexanes. First compound to elute was the
acridone label dimer 114 in 45mgyield. Increasing the eluant polarity to 60/40 eluted the
tH NMR data identical with those given in the
title compound in 10.8mg (87o) yield, with
previous procedure.
Attempted formation of 119 by reaction between triflate 113 and alkyne 69 using
1
equivalent tetrakistriphenylphosphine palladium
To a stirred solution of triflate 113 (50mg, 0.107 mmol) in DMF (2 ml) was added Pd(PPq)4
(123mg,0.107 mmol, 1 .q). A green suspension slowly formed. TLC analysis of the
reaction mixture after 2.5 hours showed the absence of riflate at Rr = 0.24 and a new spot at
&
= 0.t8 (50/50 EtOAc/hexanes). Triethylamine (0.5m1), CuI (4.1mg,0.02 mmol, 0.2 eq)
and alkyne 69 (37mg,0.107 mmol, leq) were added and stirring continued. TLC analysis of
the dark brown reaction mixture after 30 minutes showed the absence of the green
intermediate, formation of the alkyne dimer 114
(& = 0.45) and unreacted
alkyne 69 (& =
0.80). No fluorescent spot at ca \ = 0.10 corresponding to coupled product 118 was
observed.
Experimental 120
N-Benzoyl-4-t(N-biotinyl)-11-aminoundec-1-ynyll-l-phenylalanine methyt ester (120)
23
6"
5
5"
3'
.H
H-
1
Eluant 5Æ5 MeOfVCflC!; recrystallisation from MeOH; light yellow crystals mp 142";
(y4Vo)yield. HRMS Calculated for C*I!o\OrS: 674.3502. Found: 674.3520- MS:
(M*, 9), 615
17
(1
l),521 (14),
461
(L2),227 (12), 105 (100). IR (nujol): 3296br (N-H),
tH NlvIR: 1.29
42m, 1706s and, I642s (C=O), 1538s.
2.17
674
-1.7
6 (m, 22H, methylene protons);
(t,2IJ,J7.5Hz,C[!CON); 2.38(t,J 6.9Hl2, CHr-=-Ar);2.76(d,l}l,Jr l2'8IJ2,
cs-Hb); 2.86 (dd, 1H,
.4*
I2.8, 4.9 lirz, CS-IJa); 3.09-3.31 (4H, m, CHr-At and CIINHCO);
3.75 (s, 3H, CH3O2C); 4.28 (dd, l}j,',
I7 .2, 4-5 IJz, C4-H); 4.48 (dd, lH, J 7 .6, 4'9 Hz, C3-H);
5.06 (dt,LIf,.,n.4,5.8 Hz, crC-H); 5.55 (ås, lH, Nl'-H); 6.01 (r, lH, J 5.6H2, CH'-NHC=O);
6.33(bs,lH, N3'-H);6.85 (d,lIH',I7.5}Jz, C2-NHCO);7.06 (d,2H,f 8.IHz, C3-Hand
C5-H); 7 .31 (d,z1, J 8.1 Hz, Cz-Hand C6-H) ;7 .39-7 .75 (m,
5]H,
PhCONH). "C NMR:
19.4,25.7,26.9,28.!,28.2,28.7 ,28.8,29.1,29.3,29.4,29.6,36.0,37 .6,39.5,40.5,46.2,
52.5,53.5,55.6, 60.2,61.8 (alkyl), 80.2, 90.8 (alkynyl),122.9,127.1,128.6,129.2, L31.7 ,
1
3 1. 8,
L33.7, 135.4 (aryl), 1 63. 8, 166.9,
17
2.2,
17 3. 1
(carbonyl).
N-benzoyl-4-t¡f-(5-dimethylamino-l-naphthalenesulphonyl)-1l-aminoundec'l'ynyll'l'
phenylalanine methyl ester
(l2l)
23
2"3
4
6"
H
CO2Me
5
NM%
8
76
Eluant 40/60 EtOAc/hexanes; fluorescent green viscous oil;82Vo yield. Calculated for
c4¡Io7N3OrS: 681.3236. Found: 68I.3253. MS: 681 (M*, 68),649 (12),560 (22),203
(55), 169 (100), 105 (79). IR (thin film): 3300brrn (N-H), 1736s,1652s (C=O), 1578s,
1512s,1316s andl144s (O=S=O),910s,738s. UV (EIOH): 227 (36200),247 (39 600), 338
Experimental
tH
(5 700). Fluorescence @toH, 1,", = 338 nm): 503 nm.
NMR: r'30-l'21 (m,l2IJ,
merhylene protons); I.47 (quíntet,2IJ, J 7.3IJ2, CH2-CH2-NHSOT);2.29 (t,2IJ, J
Ct!--:Ar);2.79 (q,2]H,J 6.5IJ2, CHr-NHSOT);2.80
l2l
7.IIfz,
(s, 6H, (CH.)fN); 3.13 and 3.2I
(2x
dd,lIJ,,f 13.8,5.6H2,C3-H);3.69(s,3H,CIl-OrC);4.58(r, lH, f 6.5HZ,SOrM-CI!);
(d,2H,J8'2H2,
5.01(dt,lIF,.,J7.5,5.6Hz,gC-H);6.53(d,lH.,J7.SHz,CONH);6.97
c3'-H and c5'-H); 7.10 (d, lli^,
7 .32-7
.5I (m, 5H, At-rÐ;
7
I 7 .4H2, C6-H); 7 .24 (d,2}l, r 8.2H2,
.63-7 .67
(m,2}J, At-H); 8.17 (dd, lH', J 7.5,
(d,lIJ,f 8.6H2,C4-H); 8.46(d,l]g.',J7.5Hz,C2-H).
28.8,28.9,29.1,29.4,37
.7
Cz',-}l and C6'-H);
l.l Hz, c3-H); 8.21
t'C NMR: 19.3,26.3,28.6,28.7,
,43.2,45.4,52.4,53.4 (alkyt), 80.2, 90.7 (alkynyl), 115.1, 118.7,
122.9,123.1,127.0,128.3,128.6,129.2,129.2,129.6,129.8,130.3, 131.7,131.8,
13
4.7,
13
5.3, 152.0 (aryl),
1
66.
8,
17
1
133.7 ,
.9 (carbonyl).
N-Benzoyt-4-t11-(N-5'-fluoresceinyl)-11-carbonylaminoundec-1-ynyll'L'phenylalanine
methyt ester (122)
o
ï
2"
3"
4',
3'
4
H
CO2Me
5'
6
1
2
Eluanr 5Æ5 M9OIVCIJ"CIT; bright orange glass; 75Vo. IfRNÍS (LSIMS) Calculated for
CorI{orNP, (M+H.): 793.3125. Found: 793.3096. MS: 793 (M+H*, 100), 594 (19),402
(22),347 (18). IR (CDClr): 3500-3000br w (Ãr-oH), 3080w (tu-H), 1742s and 1644s
(C=O), t602s (Ar C=C). UV @tOH): 23I (39 900), 257 (31100),457 (5 600),483 (5 900).
Fluorescence @tOH, L..
=
483 nm): 518 nm. The NMR data are
for the lactone structure.
Experimental 122
tH NMR (d6-DMSO):
l.2l-I.61 (m,
L2H, methylene protons); 2.36
cr!-=-Ar); 3.08 and 3.16 (2 x dd, IH', J
13.7 ,
(dt, l}J, J 7 .9, 5.4H2, aC-H); 6.52 (dd,zIJ, J
(t,2F{,I 6.7 rlz,
5.4IJ2, CH"-Ar); 3.63 (s, 3H, CHtOTC); 4.64
8.7
, 1.9 IJ:z, C2-H); 6'58 (d,zH, J 8'7
}lz,
C1-H); 6.61 (d,2If,., J L.9 llj¡z, C4-H); 7 -17 (d,lH, -I 8.4}J2, C3'-H); 7 '26 (bs, 4}l', C2" -}l,
c3"-H, C5"-H, C6".H); 7 .4t-7 .54 (m,3IH, PhCON); 7.77 (m,2H, PhCON); 7.82 (dd, l}f, J
8.4, 1.6
1Frz, C4'-lHt);
8.33 (d, llF.,I I.6Hz, C6'-H); 8.85 (d, lH, .I'7.9Hz,PhCONH); 10.20
(bs,2H,Ar-OH); 10.40 (bs, lH,5'-NH).
t3C
NMR (d6-DMSO): 18.6, 25.O,28.2,28.3,28.4,
28.6,28.7,29.0,36.0,36.5,54.0,51.9 (alkyl), 80.5, 83.0, 90.4, L02.1,109.8, 112.8,121.5,
126.2,127.2,127.6,128.3,l2g.l,129.5,130.9, 131.3, 131.5, 133.6,I37 .6, L40.9,152.0,
1
59.9 (aryl), 166.4, 1 68.7,
17
2.0,
17
2.1 (carbonyl).
N-Benzoyt-4-(12-{5-[1,10-phenanthroline-áis-1,10-phenanthrolineruthenium(II)]dodec'1
-ynyt))phenylalanine methyl ester hexafluorophospate (123)
2"
Ha.
3"
4
z',
naz
6"
5"
2
6'
.2PF6
7
8'
3
4
9'
5
Chromatography on alumina with eluant CHCI,; bright orange glass; 657o. MS (LSIMS):
t232 (fio2RuM-PFuì*, 82), 1087 1'02RuM-2PFu, 100). UV (CHCIr): 263 (87 600)' 418 (L2
tH NMR (300 MHz, ô
000), 451 (12 S00). Fluorescence (CHClr, f,". = 451 nm): 573 nm.
ppm): I.22-l.6l,m,l4[,methyleneprotons; I.86,quintet,2H,J7.2Hz,CIL-CIt-Ar;2.38,
t,2}J,I6.9Hz,CHr-=-Ar; 3.L7-3.3l,m,4H,CHr-Ar
5.05, dt,lIFr, J 7.3,5.8 Hz, C-H; 6.58,
and
CF!-phen;3.76,,r,3H, CIIOTC;
bd,lH, "I7.3H2, PhCONH;
7.O5,
d,2IJ, J 8'0 Hz,
C3"-H and C5"-H;7.31, d,2H,,/ 8.0 Hz, Cz"-H and C6"-H;7.40-7.86,fi4Hi 7'89, s, lH,
C6-H; 8.O4, d,
4}J,
L]f^,
J 5.3 if1z, Cx-H; 8.10-8.13, nt,8}l; 8.35, d, lH, -f 8'2H2, CZ'-H; 8'45, d,
J 8.2H2, Cz-Hand C9-H;
8.5'7,
d, l}l, J 8.6 Hz, C9'-H.
Experimental
N-Benzoyl -3-({11-t6-O-(methyl)fluoresceiny[}undec-1-ynyl)-1,-tyrosine
123
methyl ester
(L24)
7
oil
H.+
PhcN
H
6',
CO2Me
2
8
1
3'
5',
6'
4',
5'
Eluant 8Æ2 MeOfVCryClz; bright orange glass; gl%o yieLd. HRMS Calculated for
corHorNOr: 793.325L Found: 793.3241. MS: 793 (M*,31),734 (100), 673 (37),601 (30),
326
(78). IR (CDCL): 3436w (N-H), L726s (C=O ester), 1644s (C=O amide), 1598s, 1516s'
UV (EIOH):
225 (72 5W),253
(36200),276 (20 600), 301 (13 900), 365 (9 200),441 (23
tH NMR:
700),460 (33 000), 489 (26 100). Fluorescence @tOH, I", = 489 nm): 521 nm.
1.35-1 .5O (m,10H, methylene protons); 1.65 (quíntet,2H, J
7.2H2, CH,CF!-=); 1.86
(quintet,2IJ, J 6.8lfir2, CH2CH2-O Ar);2.48 (t,2rI, J 7.0Hi2, Cfl-=-Ar);3.13 and3.2l (2x
dd, lH, J 14.0, 5.6Hz,Cr!-Ar); 4.O9 (t,2IJ,
r
6.5}J2, CHr-OAr); 5.05 (dt,lH, "I7.3,5.5}J2,
crH-C); 6.12(bs,1H, Ar-OH);6.49 (d,L}j,',J l.9Hz,C4-H); 6.57
c2-H); 6.65 (bd,lH,
ur
7 3Hz,PhCONH); 6.75 (dd, LH,,r 8.9, 2.4H2, C7-H); 6.86-7 .00 (m,
5H, Cl-H, C5-H, C8-H, C5"-H, C6"-H);
7
.ll
l.2ftz, C3'-H); 7 .42-7 .56 (m,3Ii,^, PhCON);
r 5.g,7.5H2,C4'-H);
(dd,I}l,f 9'7,1'9}l:2,
7
(d,
l}l, I 2.l
.68 (dt,
lIJ,I
}Jz, C2"-H); 7 '33 (dd,
7
lIl,
J 7 '3,
.5, 1.4 Hz, C5'-H);7 .73 (dd,
7.75-7.7g (m,2IF^,PhCON); 8.27 (dd,LlF^,r7.6,1.3 Hz, C6'-H).
l}l,
13C
NMR:19.6,25.9,28.4,28.9,29.0,29.1,29.2,29.3,36.9,52.4,53.7,68.9(alkyl),74.9,97.3
(alkynyl), 100.7, 105.7, llo.7 ,114.0, 114.6,114.8, ll7
.4,ln J,
127
.2,128.4, 128.6,128.8,
129.7,I29.9,130.0, 130.3, 130.3, 130.5, 131.1, 131.7,132.5,132.7,133.9,134.6,150.7,
L54.4, 156. 1, 159.0, 163.8 (aryl), 1 65.6, 1669,
17
2.0, 1 85.6 (carbonyl).
Experímental I24
N-Benzoyl-3-tN-(biotinyt)-11-aminoundec-1'ynyll-L'tyrosine methyl ester (125)
6"
corMe
5"
Eluant 8Æ2 M9OFVCH TCIT;767o; light yellow crystals mp 88-90". HRMS Calculated for
(M*, 74),63L (14),569 (12),537
QrHrNoOuS: 690.3451. Found: 690.3464. MS: 690
(33),477 (50), 413 (37), 105 (100). IR (CDCI3): 3500br w (N-H), 1706s and 1656s (C=O),
l470s. tHNMR: 1.26-1.73 (m,2}H,methyleneprotons); 2.17 (t,zH',J7.4H2, CI!-C=O);
2.45 (t,2.Fr,J 6.8lHL2, CF!-=-Ar);2.68
(d,l}J,JE
L2.8 Hz, C5-Hb); 2.86
(dd,lH,JB
t2.8,
4.8H2,C5-Ha); 3.07-3.24 (m,5H.,C2-H, CFL-NHCO and Cr!-Ar); 3.78 (s, 3H, CI{rOrC);
4.27
(dd,lH,,f 7.5,4.8H2, C4-H);4-46(dd,lIjr,I 7.5,4-9Hz' C3-H);
4'99
(dt,lIJ,I
7'5,
5.9H2,C2'-H); 5.46 (bs,lH, Nl'-H); 6.18 (r, lH, J 5.6H2, CI{r-NHCO);6'26 (bs, lH,
N3'-H); 6J4 (bs,lH, Ar-OH); 6.85 (d, lIJ, J 8-4IJ2, C5"-H); 6.92 (d,lH,
NHCOAT) ; 6.96 (dd, lIF^, J 8.4, J 2.1 H;z, C6"-H);
7
"I
7
'5 Hz,
.70 (d, l}J, J 2.1}Jz, C2"-H);
7
.40-7 .53
t'C NMR: 19.5,25.6,26-8,28.0,28.1,28.5,
(m,3H) and7.74-7.77 (m,2H, PhCONH).
28.6,28.8,29.0,29.2,29.5,35.9,36.8, 39.5, 40.5, 50.8, 52.4,53.8,55.4, 60.1, 61.8 (alkyl),
75.O,97.2(alkynyl), 110.7,114.8, 127.1,127.3,128.6,130.2,131.8, 132.5,133.8, 156.0
(aryl), L63.7, 167.1, 172.3,173.2 (carbonyl).
Ethyt 2-acetamidopent-4'ynoate (126)
AcN
H
A solution of diethyl acetamido malonate (30.19, 0.139 mol) in DMF
(50m1) was added
dropwise over 90 minutes to a cooled (ice bath) suspension of hexane washed NaH (807o in
mineral oil, 6.909, 0.173 mol, 1.25 eq). The mixture was stirred at room temperature for 30
minutes, then cooled in an ice bath and propargyl bromide (807o wt. in toluene, 24.7 9,0.166
mol, 1.2 eq) was added dropwise over 60 minutes. After stirring
at
70'overnight, TLC
analysis (50/50 EtOAc/hexanes) showed a trace of starting material at Rr 0.32 and a large spot
Experimental
125
corresponding to product at Rr 0.56. The da¡k brown reaction mixture was cooled to room
temperature, filtered to remove precipitated inorganic salts (Buchner), LiCl (5.889, 0.139
mmol, 1.0eq) and water (2.499,0.139 mmol, 1.0eq) were added and the mixture stired
ovemight at 145". After cooling to room temperature, the reaction mixture was poured into
water (500m1), the aqueous mixture extracted with CH,CI (250m1), the organic extract
washed with water (3 x 250m1), dried (MgSOJ and solvent removed. Residual DMF was
removed under vacuum (oil pump), the residue separated by squat chromatography eluant
50/50 EtOAc/hexanes and recrystallised from CFlClrlhexanes to give the title compound as
colourless needle crystals mp 7l-7?) (1it.4t 73") in7 .24g (28Vo) yield.
MS:
183 (M*, 5), 144
(22), tLO (66), 104 (59), 68 (100). IR (nujol): 33L2s (N-H), 3264s (H-C=), 1728s (C=O
tH MVÍR: 1.30 (r, 3IJ,J7.0IJ2, cHr-cHrr;2.03
(C{
1554s, 1232$
esrer), t634s
amide),
(r, lH, J 2.7 Hz,H-C=); 2.06 (s, 3H, CH3CO);2.78 (2H'd.d'
I 4.5'2.6H2, C[L-G);
2IJ, f 7.2H2,-OCH2CH3); 4.72 (dt, lH., J 7.8,4.6}J2, aCH-); 6.33
4.25 (m,
(d,lH, J 5.6Í1z,, N-H).
lrc NMR: 14.0,22.3,22.3,50.5,61.8 (alkyl),71.4,78.4 (alkynyl),169.7,170.3 (carbonyl).
Ethyl 5-(1-pyrenyl)-2-acetamidopent'4-ynoate (127)z Method A.
AcN
H
A mixture of the alkyne 126 (100mg,0.55 mmol, 1.5 eq), l-bromopyrene (102mg,0.37
mmol, 1.0 eq), Pd(PP\)4 (63mg,0.055 mmol,0.15 eq), CuI (21mg,0.011 mmol,0.3 eq)'
EqN (1ml) and DMF (2ml) was stirred overnight at 50o, at which time the catalyst had
decomposed. The solvent was removed under vacuum (oil pump), the residue separated by
flash chromatography eluant 60140 EtOAclhexanes andrecrystallised from CflC{hexanes to
give the title compound as cream crystals mp 155-156" in 63mg (44Vo) yield. HRMS
CalculatedforCrrl!,NOr: 383.1521. Found: 383.1509. MS: 383 (M*, ll)'323(71),238
(100). IR (nujol): 3320s (N-H), 1736 (C=O ester), 1644 (C=O amide). 'H NMR; 1.35 (r, J
Experímental 126
7.4IJ2, CI{r-CfL); 2.12 (s,3H, CH3-CON); 3.25 (d,2H, J 4.7 }Jz, CH,-G); 4'36 (m,2}J^,
OCHTC4); 4.95 (dt,
9H, Ar-H).
llfl^,
J 7 .5, 4.7
HLz,
aC-H); 6.57 (d, lIJ, J'l .5 Hlz, N-IÐ; 7.99-8.49 (m,
trc NMR: 14.3,23.3,24.1,5L.2,62.1 (alkyl),82.6,89.5 (atþnyl),124.4,125.1,
125.3,125.5,126.2, L26.8,127.2,I27.8,128.1,128.3, L29.7 ,130.3, 130.4,131.0, 131.1,
132.0 (aromatic), L69.9, 170.8 (carbonyl).
'When
the reaction was repeated using l-iodopyrene the yield of product was'127o-
Ethyt 5-(1-pyrenyl)-2-acetamidopent'4-ynoate (127)z Method B.
A mixture of the alkyne 125 (78mg ,0.43 mmol, 7.2 eq),l-bromopyrene (100mg, 0.36 mmol,
1.0 eq), Pd(PPh3)4 (41mg,0.036
mmol,0.1 eq), CuI (13mg,0.072mmo1,0.2 eq), PPh, (19mg,
0.072mmo1, 0.2 eq) and piperidine (10m1) was refluxed for 60 minutes, at which time
analytical TLC showed the absence of the aryl bromide. The solvent was removed under
reduced pressure and the residue separated by flash chromatography eluant 60140
EtOAc/hexanes to give the title compound as white crystals in 76mg (Sl%o) yield. After
recrystallisation from CflC{hexanes the physical data were identical with that from the
previous method.
'When the reaction was repeated using l-iodopyrene the yield was 707o.
Ethyt 5-(1-pyrenyl)-2-acetamidopentanoate (129)
AcN
H
CO2Et
A mixture of the alkyne 126 (294mg, 0.77mmol),57o Pd/C (100mg) and EtOAc (40m1) was
stirred under a hydrogen atmosphere overnight. The reaction mixture was filtered through
celite, the solvent removed and the residue recrystallised from EtOAc/hexanes to give the title
compound as white microneedle crystals mp 155-156' in278mg (947o) yield. HRMS
CalculatedforCrrllrNOr: 387.1834. Found: 387.1824. MS: 387 (M.,68),255(27),228
(32>,215 (100), I49 (22). IR (nujol): 3316s (N-H), 1748s (C=O ester), 1650s (C=O amide),
Experimental
844s. UV (EIOH):
2O5
127
(12200),234 (19 700), 256 (6 300), 266 (13 500), 278 (20200),302
(2 600), 313 (6 lCíJ,),327 (12 300), 343 (L4 800). Fluorescence (EtOH, L"'=343 nm): 375
(100), 395 (61), 415 (19). 'H
NMR:
1.19 (t, 3H', J
7.rllz, cHr-cHr); 1.75-2.05 (m,4g.,
C4-H and C5-H)i2.N (s, 3H, CHr-CO); 3.26-3-42(m,2F{, C5-H); 4'L4(q,2H,f
o-cH2-cH3); a.70 (dt'
(m,9H,Ar-H).
t3C
llF^'
1'lrlz,
r 7.7, 6.7 ljrz, C2-H); 5.98 (d, lH,,r 13 }tz, CONH); 7.82-8.26
NMR: 14.1,23.2,27.2,32.5,32.9,52.0,61.5 (alkyl),L23.3,124.7,
124.8,124.9,125.9, L26.7,127.2,127 .3,127 .5,128.6,131.4,135.9 (aryl), 169.8, 172.6
(carbonyl).
Experímental 128
Experimental Described in Chapter 3.2
Acetylation of Nucleosides: General Method.
To a cold (0') stirred solution of the nucleoside in pyridine (10mVg) was added acetic
anhydride (2
eq,.
or 3 eq. as required) dropwise over 30 minutes. The icebath was removed
and the mixture stirred overnight. After removal of solvent in vacuo the residue was
dissolved in
CHCI' washed with wator, dried (MgSOJ
and solvent removed. The residue
was purified by recrystallisation to give the protected nucleoside.
5-Iodo-3',5' -di-O-acetyldeoxyuridine (131)
Reaction of 51 (1.00 g,2.8 mmol) under standard protection conditions and recrystallisation
from EIOH gave rhe title compound as colourless crystals mp 163'(1it.133 163-164") in 1.18g
(95Vo)
yield. MS (FAB)
:
439
(M+H*, 7),413 (18), 207 (31), 115 (78), 93 (100). 'H NMR:
2.1,1 and,2.2l (s,3H, CI!C=O);2.15-2.22 (lø (obscured),
I 4.3, 5.6, 2.O lfrz,
lH, C2'-H);2.55 (ddd,lH,
JE"
C2'-H); 4.29 -4.45 (m, 3lH, C4'-H and C5'-H) ; 5.24 (dt, IH, J 6.5, 2.0 }l:z,
c3'-H); 6.30 (dd, Lili^,I8.2Hz,5.6Hz,C1'-H); 7.98 (s, lH, C6-H); 8.44 (bs,lH, N5-H).
NMR:
19.8, 20.0 (CH3C=O) ,36.6, 62.8,68.6,73.2,8I.3 (alkyl), 84'2, 142'8,
13C
149'l
(aromatic), 159.4, 169.0, 169. 1 (carbonyl).
8-Bromo-2',3' $' -tri-O-acetyladenosine (132)
Reaction of 52 (500mg, 1.4 mmol) under standard protection conditions and recrystallisation
from TIIF gave the title compound as colourless crystals mp 186-187" (litl3a. 187-188'dec.)
in 0.639 (92To)yield. MS (FAB): 472/474 (1:1, M+H*,64),389 (45),258 (100). 'H NMR:
2.05 ,
2.12 and 2.16 (3 x s, 3H, CH,C=O); a.30-4 .42 (m, 2H', C4'-H, C5'-Hb); 4'53 (dd,
C5'-Ha); 5.66 (ås, 2H, C5-NII2); 5.95 (r, lH, J 6.0Hl2, C3'-H); 6.ll
^Ll.2,3.OlFrz.
t'C NMR
J 4.3H2,C1'-H); 6.35 (dd, l1g^, J 6.0,4.31912, C2'-H);8.32 (s, lH, C4-H)'
JE
lH,
(d,lH,
(du-DMSO): 19.3,19.4,19.5 (CH3CO)' 61.6' 69.0' 70.6' 78.7,87.2 (a1ky1), 1I9.3,125.2,
149.3, 152.0, 153.9 (aryl), 168.2, 1 68.3, 1 69. 1 (carbonyl).
Experimental
129
8-Bromo-2'13'15'-tri'O-acetylguanosine (133)
Reaction of 53 (1.00 g,2.5 mmol) under standard protection conditions and recrystallisation
from acetone/water gave the title compound as colourless crystals mp 214-217' (lit.r34
lH NMR:
216-21.8")in 1.01g (82%o)yield. MS (FAB): 4881490 (1:1, M+H. ,16),259 (100).
1.72,1.77,l.7g (3 x s, 3H, CHrCO); 3.95-4.04 (m,2}J, C4'-H and c5'-Hb); 4.18 (m,lIJ,
cj'-Ha);5.58 (r, lH,,r 5.9H2,C3'-H); 5.62(d,lH,,r 43H2, Cl'-H); 5.64(bs,2H, C2-N$);
5.85 (dd,1H,
.,r
5.g, 4.3:H2, C2'-H); 10.38 (ås,
Ig.l,lg.2 (cllco),
t54.6 (C2),
167
.8,
lH, N1-H). t'C NMR (d6-DMSO): 19'0,
61.4, 68.8, 70.3,78.I,86.7 (alkyl), 116.5, 119.0, 150.5, 152.4 (aryl),
167
.9, 168.8 (carbonyl).
5-tN-(Biotinyl)-l-aminoundec-1-ynyll'3',5'-di-O'acetyl-2''deoxyuridine
rI
(134)
TI
H
To a stirred mixture of DMF (1.0 ml) and EqN (14mg, 0.14 mmol,I.2 eq) at room
temperarure were added sequentially 5-iodo-2',3'-di-O-acetyldeoxyuridine 131(50mg,0.11
mmol), the alkyne 107 (54mg, 0.14 mmol, 1.2 eq), Pd(PPh3)4 (13mg, 0.011 mmol, 0'1 eq)
and CuI (4.3mg, O.022mmol, 0.2
eq). The mixture was stirred
at room temperature
until
TLC indicated the absence of the nucleoside. The solvent was removedinvactn, the residue
separated by flash chromatography using 10/90
MeOIVCIIC!
as eluant
to give the title
compound as a colourless glass in 60mg Q67o) yield. HRMS Calculated for CroHroNrOnS
(M+H.): 704.3329. Found: 704.3325. MS (FAB): 704 (M+H*,31),504 (24),394 (100),
tH
307 (48), 227 (29).
NMR:
1.09-1.61 (m,lsE,methylene protons); 1.95 and 2.01
3H, CH3CO); 1.96-2.13 (m' 3lH, CH2CONH and C2'-Hb);2.21 (t,2H, J 7 .0}J2,
(2xs,
C\-=);2.31
(ddd,lIJ,J".^!4.3,5.9,2.4lFr2,C2'-Ha);2.58 (d,lH.,J8 l2.8 Hz, C5"-Hb);2.74(dd,I}l,
J8
12.8,5.0 Hz, C5"-Ha); 2.96-3.06 (m,3IJ, C2"-H and CIINHCO); 4.10-4.15 (m,2H,
c4'-H and c4"-H); 4.19 (d,2lri^, J 3.2H2, C5'-H); 4.3I (dd, lIJ, J 7.7,5J Hz, C3"-H); 5.08
Experimental
130
(dt,L}J,16.7,3.9H2,C3'-H); 5.42(bs,lH, Nl'-H);5.56 (ås, lH, N3'-H); 6'15 (dd,I}l,J
7.g,5.gH2, cL'-H);6.39
(bt,lll,J
5.2H2, CFTNHCO);7.54 (s, 1H, C6-H); 11.20 (ås, 1H,
N3-H). trc NMR: 18.8, 20.5, 20.7,25.3,26.4,28.0,28.2,28.3,28.5,28.8,28.9,29.2,35.2,
36.2,38.3,39.8,45.9,55.4,59.2,61.0,63.5,72.5,73.8,81.5,84.8 (alkyl),93.5,99.7
(alkynyl), 142.1 (el.yl), 149.3,
1
6
1.5, 162.7, 1 69. 8, 169 -9, 17 1.8 (carbonyl)'
Compounds 136, 137, 138, 139 and 141 were prepared using the appropriate alkyne in a
similar manner, except for the differences stated.
5-{11-t6-O-(Methyl)fluoresceinyU-l-undec'l-ynyl}'3',
5'-di-O-acetyl-2'-deoxyuridine
(136)
H
3
6
3'
5
4
I
I
at
4
6
5
Eluant 7:93 MeOH:CIlClr; bright orange glass; 767o. IfRMrS Calculated for CorH,uNrO,,
(M+H.): 807.3129. Found: 807.3115. MS (FAB):
807 (M+H., 100),747 (10),607 (22),
347 (80). IR (CDCL): 3400w (N-H), 1760-1680s (C=O), t644s,1598s. UV @tOH): 232
(66 400), 27g (29900),435 (23 000), 460 (32900),489 (25 S00). Fluorescence (EIOH)
tH
= 4g9 nm): 521 nm.
7
NMR:
1.36-1.62 (m, L2H,methylene protons); 1.85 (quintet,2}l, J
.2Hz, CHr-CFt-O -Ar); 2.14 and2.20 (2 x s, 3H, CFI3CO);2.2a (dd' l}J'
C2'-Hb); 2.4I (t,2l,i{^,f 7.lrirz, Ctt-:-Ar);2.54
(ddd,IrI,Js
3.66 (s, 3H, CtL-OrC); 4.08 (t,2IFr, J 6.6H2, CIt-OAr); 4.30
L]f.,
rs
I4.4, 6.7
}lz,
14.4,5.8 Hz, 2'4Hz,C2'-Ha);
(q,lH, J 2.9IJ2, C4'-H);
(d,2}J, J 3.0}í12, C5'-H); 5.26 (dt,lH, J 6-4H2,2.4}J2, C3'-H); 6.32 (dd,
Cl'-H); 6.47 (d,
(\.
4.38
llf, J 8'0, 5'8 Hz,
J l.9ftz, C4"-H); 6.55 (dd,lH, 'I9.7,I.9}J2,C2"-H);6'73 (dd,7}l,I
8.8,2.4IF12,C|"-lFf); 6.85 (d,lIFr,J 9.3IFr2, Cl"-H); 6.88 (d, 1H,,f 8'7 }Jz, C8"-H); 6'95 (d,
1H, "f 2.4]f¡2, C5"-H);
7
.31
(dd,lH, ,f 7 .5,I.4 Hz, C3"'-H);7 .67 (dt, l}l,I 7 .5, l'4H2,
C5"'-H); 7.72(s,lH, C4-H);7.74(dt,lH,J7.4,I.5Hz,C4"'-H);
8.23
(bs,lH, N3-H);8'25
Experimental
l3l
(dd, lIJ, J 7.7,I.2H2, C6"'-H). t'C l.IIvIR: 19.5,20.7,20.8,25.8,28.4,28.8,28.9,29.O,
29.2,29.3,38.O,52.4,63.8, 68.8, 71.0,74.O,82.4 (alkyl), 85.2, 95.4 (alkynyl), 100.6, 101.4,
105.6, 113.8, 1L4.6,117.4,128.7,L29.6,L29.7,130.1, 130.2,130.5, 131.0, 132.6,134.6.
140.5, 149.2,150.3, L54.3,158.9 (aryl), 161.4,163.6, 165.6,170.0, 170.3,185.6 (carbonyl).
S-t11-(10-(1011)-g-Acridonyl)undec-l-yn Yll'2',3',5'-tri'O-acetyladenosine (137)
AcO
Reaction at 50' f.or24hours; Eluant 50/50 EtOAc/hexanes; pale green glass; 897o. HRMS
(LSIMS) Calculated for CootlorNuO, (M+H.): 737.3299. Found: 737.3295. MS: 737
(M+H*, 54), 479 (100), 208 (23). IR (nujol): 33l2br m,3I64br m,2232w, 1748s, 1634s,
1600s, 1496s,1232s. UV (EIOH): 218 (30 700), 234 (3O lO0),257 (46 700)' 293 (17 800),
386 (7 060), 405 (7 S30). Fluorescence @tOH, f,". = 386
NMR: l.4¡-1.6t (m, 10H, methylene protons);
nm): 418 (100), 439 (70). lH
1.73 (quintet,2E, J 7 -IIfz, CH2CF!-:); I.97
2.ll and 2.15 (3 x s, 3H, CHrCO); 2.58 (t, 2Il, J
'l.lIJz, CF!-=-Ar); 4.31-4.43 (m,H,); 4.54 (dd, lH,I 11.4,3.3Hl2,); 5'73 (bs,2H,
(quíntet,
zIF,^,
r
7
.9 lFrz, CHTCH2-N); 2.06,
C2-NH'); 5.99 (m,lH, C3'-H);6.24-6.27 (m,2H, Cl'-H and C2'-H);7.32(dd,zIJ,J 8.7,7.8
]Jz, C2"-H);7 .52
(d,2H,I
8.7 lfliz, C4"-H);
7j5
(ddd,2}l, J 8.7,6.9,I.5}Ji2, C3"-H); 8.37 (s,
t'CNMR: 19.54,20.46,20.54,20.70,
lH, C4-H);8.61 (dd,2R,J7.8,1.5r{r2,Cl"-H).
26.91,27.20,27.86,28.88,28.98,29.32,29.40,46.75,63.12,69.70,70.43,7251,79.73,
87.32,98.91, 114.5I,L2I.19,122.46,128.01, 133.88, 134.77,141.74,149.24,153.76,
155.00, 169.28, 169.46, 170.60, r77 .98.
Experimental I32
8-tN-(Biotinyl)-1l-aminoundec-l-ynyll-2'n3'. ,S'.-tri-O-acetyladenosine (138)
H
Eluant 10/90 MeOfVCflC!; recrystallisation from EtOAc/hexanes; pale yellow crystals mp
88-90'; 88Vo. Calculated for CrrH'NrOrS (M+H.): 785.3656. Found: 785.3680. MS
(FAB): 785 (M+H. ,62),527 (100), 301 (12), 259 (13). IR (CDCL): 3500u 1740s,1708s,
1632s, 1470s.
tH NMR: 1.23-1.75 (m,20H, methylene protons); 2.04,2.10 and2-13 (3 x s,
3H, CH3CO);2.I7 (t,2lH,J7.3lHL2, CrtcoNH);2.54 (t,2H.,J 6.9Id2, Ct!-=-Ar);2.74 (d,
1H,
/r*,
12.7 lHz, CS"-Hb); 2.89
(dd,1H,,fs* 12.7, 4.8 Hz, C5"-Ha);3.14 (dt, lIJ, J 6'0, 4'6
IJz, C2"-Ff); 3.19 (q,2lfir, J 6.6IgL2, CHTNHCO); 4.28-4.a0 (m,3II, C4'-H, C5b'-H and
C4"-H); 4.5t (m,zif',Cs'-Ha and C3"-H); 5.95 (t,lH,,f 5-5}ll2, C3'-H); 6'08 (bs'
Nl"-H); 6.17 (bt,1H,./
5.4lFr2, CHTNHCO);6.22 (m,2}J,
Cl'-H
and
lH'
C2'-H);6.33 (bs,2Il,
C2-Nf!); 6.52 (bs,1H, N3"-H); 8.30 (s, 1H, C4-H). "C NMR (75lvlflz, ô ppm): 19.46,
20.43,20.51,20.67,25.60,26.80,
.76,28.06, 28.19,28.74,28.85, 29.10,29.21,29.51,
27
36.02,39.40,40.56,55.62,60.16, 61.85, 63.08' 69.62,70.40,72.39,79.69 (atkyl)' 87 '28,
99.00 (alkynyl), llg.25, 134.54,149.05; t53.69,155.26 (aryl), l&'22,169'30'
169.46,17 0.60, l7 3.07 (carbonYl).
S-U
1-
(139)
t6-O-(Methyt)fl uoresceinyll- 1-undec-1-ynyl)-tri-O-acetylguanosine
'\
J
4
.}
AcO
3',
3
T
I
4
4
5
7
2
8
1
2
4
6
5
Reaction at 50'for 5 hours; eluant 7.5192.5 MeOFVCFICI; recrystallisation from MeOH;
bright orange glass; 517o. HRMS (LSIMS) Calculated for CorHroNrO,, (M+H*): 904.3405.
Experímental 133
Found: 904.3411. MS: 904 (M+H., 100), &6 (32),347 (88), 259 (27). IR (CDClr):
3t32m(N-H), 1748s,1720s,1688s (C=O), 1644s,1596s. UV (EIOH) À-"' (e): 230 (25200),
27g (t8400), 363 (3 900), 437sh(9 600),460 (13 600),489 (10 800). Fluorescence @toH,
I". = 4g9 nm): 522nm. lH NMR:
2IJ, J 7.2}ã2,
1.26-1.48
(m,lDIl, methylene protons);
1.65 (quintet,
CHr-Ct!-=); 1.84 (quintet,z]fl, J 7.1, CHz-Cry-OAr); 2.05,2.11 and 2.12 (3 x
s,3H, CFLCO); 2.5L (t,zE,J 6.9Hz,CF!-=-Ar);3-65 (s,3H, C[-OAr); 432(m,2H,C4'-H
and C5'-Hb);
a.5l (m,lIF., C5'-Ha); 5.88 (br s,ZIF., C2-NI!); 5.93 (t, LIJ,J 5.8 Hz, C3'-H);
6.09 (m,2H, Cl'-H and c2'-H); 6.39 (d,
LIF,',
J
1.5 IJz, C4-H); 6.48 (dd,
lH, J 9.8,
C2-H): 6.76 (dd,lH, ,f 9.0,2.2H2, C|-IJ); 6.86 (d, IIJ, J 7.7 Hlz, Cl-H); 6'89
Hz, C8-H); 6.99 (d,
lIF^,
f 23H2, C5-H); 7 .34 (d, I}J, J 7.3IJ2,
IIz, C5"-H);7.78 (r,
1H,
f
Nl-H).
t3C
Hz,
1.5
(d,I}l,
J
7
'l
C3"-H); 7 '70 (t,I}J, J 7 '0
6.9Ii,¡2, C4"-H); 8.25 (d, lIJ, J 7.8}J:2, C6"-H); 10'69 (bs,
lH'
NMR (dó-DMSO): 19.2,20.4,20.4,20.6,25.7,27.8,28.6,28.7,28.8,29.0,29.2,
52.2,53.7,62.9,68.8,70.0,70.2,72.1,78.!,79.2,87.0,96.3,
100.7,IO5.2,113.8, IL4.4,
!17.0,117.4,128.8, 129.7,129.9,130.3, 130.4, 131.0, 132.8,134.3,150.8, 153.8, 154.1,
156.6, 158.9, 163.7,165.4,169.1, L69.3,170.4,185.1. Also recovered was the diyne 140 in
tH
9.3mg (l2To) yield.
NMR:
1.25-1.47 (m, l2H, methylene protons); I.45 (quíntet,2}l, J
6.8Hz,CHrCH2-O Ar);2.t7 (t,2H,J 6.8IJ2, CII2-:); 3.56 (s, 3H, CII3O2C); 3.98 (t,2H,J
6.5Hz,Cf!-OAr); 6.39 (d,lH,.I l.9Hz,C4-H); 6.47 (dd,lII,J9.6,l'9F{z,C2-H);6'65
(dd,l}i^,J8.7,2.3Hz,CY-g); 6.77 (d,!H,f 93Hz,C1-H);6.80(d, lH,'f 8'7H2, C8-H);
6.87
(d,lH,
.r
23H2, C5-H); 7 .23 (dd,lH,
.r 7.4,
l.zHz, C3'-H);7
.60 (dt, l}J, J 6.2,
C5'-H); 7 .67 (dt,1H, ,f 7 .4, !.4H2, C4'-lI); 8.17 (dd, lH., .I 7 -6, I.2Hz, C6'-H)'
4
5
1
8
2
7
3',
4
6
5'
t40
l.4Hz,
Experimental 134
8-tN-(Biotinyl)-1l-aminoundec-1-ynyll-2"3"5'-tri-O-acetylguanosine
(141)
o
o
H
-H
Reaction at 50'for 6 hours; Eluant 10/90 MeOIVCIIC!; recrystallisation from MeOH; pale
yellow crystals mp 157-160";58Vo. HRMS (LSIMS) Calculated for CrrIlrNrO,oS (M+H*):
801.3605. Found: 801.3597. MS: 801 (M+H*, 65),543(29),261(100). 'HNMR
(cDC\td6-DMSO): 1.20-1.58 (m,20E,methylene protons); t.99,2.05,2.09
(3 x s, 3H,
cHrco); 2.03 (t, zlFr, J 7 .8 lFrz, CILCONH); 2.53 (t, 2IH, Ct!-=-Ar) ; 2.64 (d, lld, J E
Hz, C5"-Hb);2.80
(dd,llH,Js l2.3,5.lHz,C5"-Ha);
(q,2IJ, J 7.2H2, CH2NHCO); a.09-4.31 (m,
lH,,r I1.7,3.8H2,
lfi,.,
2.99
12.3
(q,lH,,r 6.2H2, C2"-H);3.06
C3"-H, C4"-H, Cs'-Hb, C4'-H); 4.41 (dd'
CS'-Ha); 5.58 (r, lH, J 6.0Hl2, C3'-H); 5.97 (m,2H, Cl'-H and C2'-H);
6.35 (bs,lH, Nl"-H); 6.42 (ås, lH, N3"-H); 6.73 (bs,2H, C2-NI{2);7.74 (bt' lIJ, J 5.5 Hz,
CHTNHCO); 10.97 (ås, lH,
27
Nl-H). "C NMR (d6-DMSO):
8.5 18.50, 20.2,20.4,25.3,26.4,
.5,28.0,28.2,28.5,28.7 ,28.9,29.2,35.2,38.4, 45.5, 55.5, 59.2,61.0, 62.8, 70.2,78.9,
96.3, rL6.4, L29.3. 150.6, L54.2, 156.0, 162.7, 169.3, 169.4, 170. 1,
17 1.8.
Experimental
135
Experimental Described in Chapter 3.3.
17
-ll2-(l-Pyrenyl)dodeca-
1,1
1-diynyu-3-O-methylestr-16-ene (144)
To a stfured mixture of DMF (1.0 ml) and EqN (0.2 ml) was added sequentially the riflate 54
(50mg,0.12 mmol), alkyne 74 (52mg,0.14mmo1, l.2eq),Pd(PPq)4 (14mg,0.012 mmol,0.1
eq) and CuI (4.6mg ,0.024 mmol, 0.2 eq). The reaction mixture was stilred at room
temperature for 3 hours, at which time TLC
Q\nO CH"Clrlhexanes) indicated the absence of
the triflate. The solvent was remov ed in vacuo and the residue subjected to flash
chromatography using 30n0 CHrClr/hexanes as eluant to give the title compound as a viscous
oilin 69mg (9Zfto)yield. HRMS CalculatedforCorHorO:
MS: 628 (M*, 100),239 (S0). IR (thin film):
(c=c),
304Om
628.3705. Found: 628-3700.
(tu-H),2250and2216w (GC)'
1502s, L256s,908s, 846s,734s. UV (CHClr): 25I (29 400),265
(ll9m),275
1610s
(23
900), 295 (29700) 3t6sh (4 800), 33I (12 400), 348 (21 600), 365 (23 700). Fluorescence
lH NMR: 0.84 (s, 3H, CHr-);
(CHCI3, f,". = 365 nm): 388 (100), 396sh(75),4O7 (80).
L.37-2.3I (many m, methylene and methine protons); 2.38 (t,2IJ, J 6.6H2, CF!-=-HC:);
2.65 (t,2H,J7.0Hz,CF!-=-Ar);2.85 (m,2Pr, CFL-CH=);3.74 (s,3H, CI{r-OAr);5'91 (r,
IIJ, J l.2lfirz,HC=); 6.61 (d,lH, J 2.6H2, C4-H); 6-65 (dd, lFI, J 8.4,2.6H2, C2-H);7 '15
(d,lIJ, r 8.4H2,C1-H). t'C NMR: 16.2,19.7,20.0,26.6,27.8,28.8,29.0,29.0,29.r,29.1,
29.2,29.5,3L.6,34J ,37 .6, 44.3, 48.1,55.2,55.3 (alkyl),76.1,77 .6,79.7 ,93.9 (alkynyl),
96.4,11I.4,113.9,118.9,124.5,125.3,125.4,I25.7,126.1,126.1,127 .3,127
129.6, 130.7,
13
1.2, 13 1.3,
1
3
1.9, 132.9, 133.6
137 .9
l,
1
3
8.0, 157 .4 (aryl).
.7
,128.0,
Experímental L36
Compounds 145, 146 and L47 wercprepared in a similar manner as that described for
compound 144, apartfrom the differences stated.
17-
(145)
tN-(Biotinyl)-1 l-aminoundec- l-ynyu -3-O-methytestr- 16-ene
o
5
2
I
3
I
-H
4
Reaction of compound54 (50mg,0.12mmol) with alkynel07 (57mg,0.14mmo1, l.2eq)
under standard conditions and purification by flash chromatography using 6/94
MeOIVC¡IC!
as eluanr gave the
title compound as colourless crystals mp 150-152" in 70mg
(897o)yield. HRMS CalculatedforCootlrrNrOrs: 659.4121. Found: 659.4087.
MS:
659
(M*,29),644(M.-CI1,40),459 (15),416(100). 'HNMR: 0.86(s,3H'CHr-); I.22-2-35
(many rn, methylene and methine protons); 2.20 (t,2}J, J 7 .5 IJz, CFI-CONH); 2.36 (t,2}J,
6.9IJ2, CrL-=); 2.73
(d,lH,.r
L2.9IFrz, C5-Hb);
2.91(m,3H, C5-Ha and CFI-G=);3.16 (dt,
(q,2IFr, J 6.5 ljrz,
C[L-NH); 3.79 (s, 3H, CIü-O Ar); 4'31 (dd'
1H, ,f
7
3, 4.5 IFrz, C2-H);3.23
lH,
7
.5,
"f
f
4j Hz, C4-H); 4.52 (, dd, IH', J 7 .5, 4.8IJ2, C3-H); 5.21 (br s, lH, Nl'-H); 5'93
(br t, l}J, f 5.7 Hz,NHCO); 5.93 (m,lH, HC=); 6.03 (br s, lH, N3'-H); 6'64 (d,lIJ, J 2'7
t'C NMR
Hz, C4"-H); 6.72 (dd, I:g^, f 8.6,2.7 Hz,C2"-H); 7 .2L (d, lI¡¡,I8.6 Hz, Cl"-H)'
(d6-DMSO): 16.0' I8.7'25.2,25.3,26.0,26.4,27.2,28.0,28.1,28.2,28.3,28.4,28.7,289,
29.1,29.2,31.1,34.2,35.2,37.1,38.3,43.7, 47.6,54.8,54.8, 55.4,59.2,61.0 (alkyl), 76.0,
93.7 (alkynyl), 111.4, 113.4,125.8,132.0,133.2, 137.2, 137.3,157.0 (vinyl and aryl), 162.6,
171.7 (carbonyl).
Experimental
L37
pyrenyl)dodec-1'ynyll-17p'acetyloxyandrost'2'ene (146)
3- t12-(1-
Reaction of triflate 55 (mixture
of
L-2 and Â-3 isomers, ca. 5:I ratio) (50mg, 0.1lmmol) with
alkyne 78 (48mg, 0.13 mmol,l.2eq) under standard conditions and purification by flash
chromatography eluant 40160 CÉlClrlhexanes gave the title compound as a fluorescent light
green viscous
oil in 65mg (85Vo) yield. HRMS Calculated for
CooHuoOr: 680.4593. Found:
680.4611. MS: 680 (M*, 76), 620 (87),326 (69),215 (100). IR (thin film): 3040m (AI-H)'
1732s
(C=O), 1446m,1250s, 1030s, 844s,740s. UV (CHClr): 248 (35 000)' 258 (11 800)'
269 (22 400),279 (35 300), 317 (10 300), 330 (21300), 346 (26
600). Fluorescence (CHCI3,
tH NMR: 0.73 and O.76 (2 x s, 3H, CI{r-);
L"*= 346nm): 379 (100), 397 (67), 4t8sh (22).
0.56-2.19 (many m, methylene and methine protons); 2.05 (s, 3H, CI{3COr);2.30
6.9 }Jz,
Crt-c=);
(d,lIJ,f
3.34 (t,2lflr, J 7 .8IJ2, CHr-Ar); 4.57
(dd,lH,
"r
9.I,7
.8 Hz,
(t,2H,J
CH-OAç); 5.91
t'CNMR: 11.8, 12.0, 12.L,19.3,20.4,
4.9Hz,HC=);7.86-8.30 (m,9H,Ar-H).
21..2,23.4,27.4,28.1,28.8,28.9,29.1,29.4,29.5,29.8,31.0, 31.3, 33.6,34.0,34.1,34.4,
35.2,36.8, 40.1, 41.2,42.4,50.5,53.5 (alkyl), 81.8, 82.8, 87.5, 119.7 (alkynyl and vinyl),
123.5,124.6,124.7,125.0, L25.7,126.4,127 .0,I27.2,127.5,128.6,129.6,130.9, 131.4,
131.8,
137
.2,137.3 (aryl), 111.2 (carbonyl).
3-tN-(Biotinyl)-11-aminoundec-1-ynytl-17p-acetyloxyandrost-2-ene
1
3' ,H
,,
5
1"
2
H
4
(147)
Experimental
Reaction of triflate 55 (mixture
of
138
L-2 and Â-3 isomers, ca. 5:L ratio) (50mg, 0.11 mmol)
with alkyne 107 (51mg, 0.13 mmol ,l.2eq) under standard conditions, purification by flash
gave the title
chromatography eluant 5lg5 MeOIVCIIC! and recrystallisation from MeOH
for
compound as colourless crysrals mp 162-164" in 76mg (887o) yield. HRMS Calculated
cofl*Nroos: 707.4696. Found:
707.4696. MS: 707 (M*, 18),632(26)'354(1L00),227
(2g),147 (27). IR (nujol mull): 3296brn (N-H), 1738m and 1704s (C=O), 1642s (urea
C{),
1246s.
tH NMR: 0.75 and 0.75 (2x s, 3H, CHr-); 0.65-2.20 (many rn, methylene and
(t,2H,
methine protons; 2.04 (s,3H, CH3CO);2.18 (t,2lFr, J 7 .3 IJz, CI{TCONH);2.28
I 7 '0
(dt'
lflz,CÍ1"-=);2.74 (d,l]f^,J l2.8Hz, CS-Hb);2.93 (dd, LIJ^,J 12.8,5.0 Hz, CS-Ha); 3.16
lH,
.r
7
.2, 4.g Hz, C2-H);3.22 (q,2lli{^, J 6.5 Hlz, CIIr-NHCO); 4'33 (dd,
c3-H); 4.52 (dd,1H,
lH, Nl'-H); 5.6a
"r
7.4,5.0lH2,
C4-]gI); 4.58 (dd,l]H5 J 9.1,7
(s, HC= Å-3 isomer);5.73 (br
lH' J 7 '4'
4'9
Hz'
.8rI2, CH-OAc); 5.07 (br s,
t,lPr, J 5.5H:2, NHCO); 5.84 (br s, lH,
ttc NMR: 11.8, 11.9,19.2,20-4,21-L,
N3'-H); 5.91 (br d,I]fI^, J 4.8Hz,HC=, Â-2 isomer).
23.4,25.6,26.9,27.4,27.7,28.0,28.1, 28.8, 28.9,29.0,29.2,29.3,29.6,31.I,34.1,34.4,
(alkyl), 81.7, 82.8,
35.3,36.0,36.8, 39.5, 40.2,40.4,41..3,42.4,50.6, 53.6, 55.5, 60.1, 61.7
87.4, L31.8 (alkynyl and vinyl), 163.8,
l7l.l,
L73.0 (carbonyl)'
Experimental
139
Experimental Described in Chapter 4.
B
iotin-N- (1-hexadecyl) -amide (150)
H
-(cHt15cH3
3'Ñ
o<'
1',N
H
1
5
To a stired solution of biotin-NHS ester (106) (100mg,0.29 mmol) in DMF (2ml) was added
l-hexadecylamine (71mg, 0.29 mmol). A white precipitate formed within 10 minutes. The
mixture was stirred overnight, the solvent removed ín vaan (oil pump), the residue purified
by flash chromatography using 10/90 MeOIVCIIC!
as eluant,
rccrystallised from MeOH and
dried under vacuum to give the title compound as a colourless solid in 115mg (837o) mp
196-198'. HRMS: Calculated for CruHorNrOrS: 467.3544. Found: 467.3536- MS: 467
(M*, 34), 450 (22), 407 (44), 166 (29),97 (37),43 (100). 'H NMR: 0.88 (¿ 3¡3, J 6.5l¡2,
CHr-CFL); I.25-I.77 (ln, methylene protons); 2.2O (t,2}J,J 6-2H2, CI{r-CON):2.74 (d, 1H,
JE !2.8H2, C5-Hb);293(dd,lIJ,JE
12.8,5.0Hz, CS-Ha); 3.L7 (dt,lH,.I7-3'4.7H2,
cZ-H);3.23 (q,2IH, r 6.6lPr2, CHr-NHCO); 4.33 (dd, l}J, J 7.7, 5.0}{2, Ca-H); 4.52 (dd,
J 7.7 IJ2,4.7
-NHCO).
lFrz,
t3C
[}l,
C3-H); 4.91 (bs, lH, Nl'-H); 5-63 (ås, lH, N3'-H); 5'65 (bt' lH, J 5'3IJ2,
1CD,OD,323K): r4.2,23.6,26.8,28.0,29.5,29.7,30-3,30.4,30.6, 30.6,
32.9, 36.9,40.5, 41.0, 56.9, 61.7,63.5 (alkyl), 175.9, 184.9.
Compounds 151, 152,153,154 and 155 were prepared in a similar manner, varying only in
the amine used, product yield and recrystallisation solvent.
Biotin-N- (1-dodecyl)-amide (151)
Recrystallised from MeOH. Recovered 86mg (7I7o) of colourless amorphous solid mp
193-196" (lir.ss194-19S). HRMS Calculated for CrrHo,NrOrS: 4II.2919. Found: 411.2904.
MS: 411 (M*, 13), 351 (100), 227 (68),186 (61),
166 (77),148
(90). 'H (CDCI3/d6-DMSO):
0.89 (r, 3H, J 6.4Hi2, CHr-CFt); 0.96-1.47 (n, methylene protons); 1.89, t,2}J, J 7.4H2,
Experimental L40
cI{rcoNH);
z.q+ (d,
LlFr,
rE L2.7 Hz, C5-Hb); 2.6O (dd, LH', Js
2.82-2.gO (m,3lFr, CTLCON and C2-H); 3.97
I2.7, 4.9 Hz,C5-Ha);
(dd,lH, .I 7 .8, 4.9 }Jz, C4-H); 4.45 (dd, lH, J
7.8}J2,4.8I12,C3-H); 5.53 (ås, lH, Nl'-H); 5.56 (bs, lH, N3'-H); 6.51(bt,lH,
-NHCO).
13C
"r
5.2Lfz,
1do-DMSO): 14.0, 22.1,25.4,26.5,28.1,28.2,28.7,28.8,29.1,29.2,31.3,
35.3, 38.6, 40.7, 55.8, 59.2, 61. 1 (alkyl), 162.7,
17 1. 8
(carbonyl)'
Biotin-N-(1-undecyl)-amide (I52)
Recrystallised from MeOH. Recovered 65mg (56Vo) of colourless amorphous solid mp
183-186'. HRMS Calculated for C,H.rNrOrS: 397.2763. Found: 397.2758. MS: 397 (M*,
to),337 (100), 226 (49),172 (55),166 (75),97 (72). 'H NMR: 0.89 (r, 3rt,r 6.4H4
cHr-crL, ; 1.26-I.77
JE
(m, methylene proton s); 2.21 (t, 2IJ,
r 7 .5 Hlz, C[L-CON) ; 2.7 4 (d, tH,
l2.8Hz, C5-Hb);2.93 (dd, !H,Js,"nI2.8, 4.9H:2, CS-Ha); 3.16 (dt,lH,
C2-H); 3.L6 (dt, lH, "f 7 3, 4.8 Hz, CZ-H); 3.23 (q,2H,
J 7 .6, 4.6lfl2, C4-H); 4.52 (dd, I}jr,
lld,I
I
6-3 Hlz,
,f
7
3,4.8H2,
CHr-NHCO); a33 (dd, LIJ,
r 7 .6H2,4.9 Hlz, C3-H); 5.16 (bs, lH, Nl'-H);
5.77 (bt,
t'C NMR: 14.I,22.7 ,25.4,25.6,27.0,28.0,
5.4IJ2, -NHCO); 5.98 (Ds, lH, N3'-H).
28.1,29.3,29.6,29.1,31.9, 36.0, 39.6,40.5,55.4,60.2,61.8 (atkyl),163.7, 173.0 (carbonyl).
B
iotin-N-(1-octyl)-amide (153)
Recrystallised from MeOWwater. Recovered 70mg (677o) of colourless amorphous solid mp
lg3-tg6". HRMS CalculatedforC,rHrrNrOrs: 355.2293. Found: 355.2293. MS: 355 (M.,
7),338 (5), 311 (g),295 (100), 227 (30),184 (56), 166 (55), 130 (68), 100(64). 'H NMR:
0.8S (r, 3lfi^, f 6.6H2, CI{r-CfL); 1.28-I.77 (ln, 18H, methylene protons); 2.20, t,2H, J 7 .3
}Jz, CÍL"-CON); 2.74 (d,lIF^,
3.17
(dt,lH,
"r
JE
l2.8 Hz, C5-Hb);293 (dd, lIJ,
JE
l2.8, 4.9}J2, CS-Ha);
7.2,4.7 ljrz, C2-H);3.23 (q,2H, J 7.lHz, CÉIr-NHCO);4.34 (dd, LH,
'17 '7,
4.9H2,C4-H); 4.53 (dd,lH, J 7 .7 Ijrz, 4.7 IJLz, C3-H); 4.92 (bs,lH, Nl'-H);5'63 (bs,ZH,
t,C NMR: 14.1,22.6,25.6,26.9,28.1,29.3,29.5,29.6,31.7,31.8,
N3'-H and -NHCO).
36.0, 39.6, 40.5, 55.4, 60. 1, 6 1. I (carbonyl), 163.4, 173.0 (carbonyl)'
Experimental
B
l4I
iotin-N-(1-hexyt)-amide (154)
Recrystallised from MeOH. Recovered 63mg (667o) of colourless morphous solid mp
183-186'. HRMS CalculatedforC,.t!r\OrS: 327.1980. Found: 327-L971. MS: 327 (M*,
2),3L0(3),283(4),267 (40), 184(68),
166
lH
(23),156(28),143(26),116(22), 100(100).
NMR: 0.89 (r, 3H, J 6.7 Hz, Ctt -CfL); 1.29-1.80 (m,lfiH,methylene protons); 2.20 (t,zH, J
7.4Hz,C[L-CON) ; 2.7 4 (d,
CS-Ha); 3.17
lIH', J
s
l2.9Hz, CS-Hb); 2.93 (dd, LId, f E l2'9, 5'0 Hz,
(dt,lIH,f 7.1,4.6ljr2, CZ-H);3.23 (q,2H,J 7.OHz, CHr-NHCO);4'34
(dd,
7.6,5.0lfl2, C4-H);4.45 (dd, IIJ,J 7.6}J2,4.6HL2, C3-H); 4.85 (ås, lH, Nl'-H); 5.54
t'C NMR (dó-DMSO): 13.8, 22.0,25.2,
(bs, lH, N3'-H); 5.63 (bt,lH,,r 5.2H2,-NHCO).
lH,
.r
26.0,27.9,28.1,29.0,30.9, 35.1, 38.2,38.4,55.3, 59.1, 60.9 (alkyl), L62.6,171.6 (carbonyl).
Biotin-N-(1-propyt)-amide (155)
Recrystallised from warer to give a colourless amorphous solid mp 193-194'(lit.8?b 195-200')
in61mg (737o)yield. HRMS CalculatedforC,rHrNrOrS: 285.1511. Found: 285.1511.
MS: 285 (M*, 4), 225 (67),166 (26),142 (45),114 (39), 100 (100). 'H NMR:
CH3-CrL-); 1.41-1.83 (llr, 8H, methylene protons); 2.21
O.92
(t,3ll,
(t,zIJ,f 7-4H2, C[L-CONH);2.75
(d, lIJ, Js, l2.g Hz, Cs-Hb); 2.53 (dd, lIJ, J 8 12.9,5.0 Hz, CS-HaX 3.L6 (dt, lH, .I 7 .4, 4'8
IJz, C2-H);3.21 (q,2IF,', J 7 .OHz, CHr-NHCO);
34 (dd, l}J,
lH, J 7.6lFr2,4.8lflr2, C3-H); 5.L2 (bs,lH, Nl'-H);5.72 (bt,
r
7
.6,5.0 Hz, C4-H); 4.45 (dd,
LIJ, J 5.2IJ2, -NHCO); 5.83 (ås'
lH,N3'-H). trcNMR(d6-DMSO):11.7,23.6,27.0,29.5,29.8,36.9,41.0,42.2,47.8,57.0,
61.7,63.4 (alkyl), 166.1, 176.0 (carbonyl).
Biotin hexadecan-l-ol ester (156)
A mixture of biotin (100mg, 0.41 mmol), hexadecan-l-ol (496mg, 2.05 rnmol, 5 eq).
p-toluenesulphonic acid (8mg, 0.04 mmol, 0.1 eq.) and toluene (5ml) was stired at reflux for
48 hours. The reaction mixtue was cooled to loom temperature, uffeacted biotin removed
by filration, solvent removed in vacuo and the residue purified by flash chromatography,
eluanr
MeOfVCflClr1:91,to give the title compound as a colourless solid in72mg
(387o)
yieldmp 113-118". HRMS CalculatedforCruHorNrOrS: 468.3386. Found: 468.3369. MS:
Experimental 142
468 (M., 100),296 (20),227 (22),97
(2Ð. THNMR: 0.90 (r,3¡¿,J 6.4¡¡2, CHr-Gt);
1.28-1.77 (nr, methyleneprotons);2.35 (t,zIJ^,J7.2Hz,Cf!-COr);
C5-HbX 2.g5 (dd,lH, Js". 12.8, 5.0 Hz, C5-Ha); 3.19
2IJ, f
(dt,lE,.f
2-76(d,lH,.4* l2'8H2,
8.0, 4.7 }Iz, C2-H); 4'08 (r'
6.8Hz,Cf!-OrC); 4.34 (dd,lH,,f 7 .5,4.5IJ2, C4-H);4'54 (dd, l}J,J
7 '5' 5'0
Hz'
t3C
l.rüvlR: 14.1,22.7,24.8,25.9,28.2,
C3-H); 4.88 (bs, lH, Nl'-H); 5.18 (ås, lH, N3'-H).
28.3,28.6,29.3,29.4,29.5,29.6,29.6,29.7,31.9,33.9,40.5,55.4,60.1, 61.9, 64.6 (aþl),
163.4,
17
3.8 (carbonYl).
Biotin esters 157, 158, 159 and 160 were prepared in a similar manner, varying only in the
alcohol used and yield obtained..
Biotin dodecan-l-ol ester (157)
Recovered in 136mg (8O7o) yield as a colourless amorphous solid mp 117-118". HRMS
Calculated for CrrHooNrOrS: 412.2760. Found: 4L2.276I.
lH NIyIR: 0.89 (r, 3H,J 6.6¡12,
(40), 166 (100), 143 (16).
MS: 412 (M*, 4), 352 (15),227
C4-); I.27-1.76
(m, methylene
protons); 2.34 (t,zlfl^, J 7 .6lH2, CIL-CONH);2.75 (d, tIJ, J 12.8 Hz, C5-Hb);2.93 (dd, lH, J
I2.8,4.gH2, C5-Ha);3.17 (dt,LlFr,J7.9,4.8H2,C2-H);4.06 (r, zIJ,r 6.8lfz, cl!-Orc);
4j2
(d¿,
LlH, J
7.4,5.0H2, C3-H); 4.52 (dd,l}j., J 7.4,5.0 Hz, Ca-H); 5.15 (bs, lH, Nl'-H);
t,C
5.51 (ås, lH, N3'-H).
NMR: 14.!,22.7,24.8,25.9,28.2,28.3,28.6,29.2,29.3,29.5,
29.6, 29.6, 31.9, 33.9, 40.6, 55.4, 60.2, 61.9, 64.6 (alkyl), 1 65.0,
17
3.8 (carbonyl).
Biotin-undecan-1-ol ester (158)
Recovered in 129mg QgVo) yield as a colourless amorphous solid mp 110-113'. HRMS:
Calculated for
(M*, 5), 338 (24),227
Ç,tI.*NrOrS: 398.2603. Found: 398.2584. MS: 398
(24),t66(100), 97 (46).'HNMR: 0.91 (3H, J 6.4Hl2, CH3-CFI2); 1'.29-1.77
protons); 2.36 (t,zlF,., J 7.3IPr2, CHr-COr); 2.76
J
s
(rn, methylene
(d,lH, Js* I2.8Hz, C5-Hb); 2.96 (dd,lH,
I2.8, 4.9 Hz,C5-Ha); 3.19 (dt, 1H, -I 7 .6, 4.9 þ¡z, C2-}l); 4'09 (t, 2}l, J 6'8 }l¡z,
cr!-orc); 4.35 (dd, lIFr, J 6.6,4.6 Hz, C4-H); 4.52 (dd, l}J, J 6.6, 4.9IJ2,
C3-H); 4.66 (bs,
Experímental 143
LH,
t'C NMR: 14.1,22.6,24.8,25.9,28.2,28.3,28.6,29.2,
Nl'-H); 4.88 (bs, lH, N3'-H).
2g.3,29.5,29.5,31.8,33.9,40.5,55.4,60.1,61.9,64.5,(alkyl),
163.7,I73.8 (carbonyl).
Biotin octan-l-ol ester (159)
Recovered L47mg (9BVo) as a colorless amorphous solid mp 121'. HRMS Calculated for
c,rIlrNPrS:
356.2134. Found: 356.2128. MS: 356 (M., 100),329 (26),287 (5I),227
(52),166 (82),97 (71). tH NMR: 0.89 (r, 3g',
methylene protons);
2j5
(t,
2'Fr, J 7
f
6.5¡a;2, CHr-CÉL); 1.28-1.76 (¡7, 18H,
.2IH2, Cf!-COr); 2.7 4 (d, lH, 'fs." 12.8 Hlz, C5-Hb);2'92
(dd,7JJ,Js 12.8,4.7Hz,C5-Ha); 3.16(dt,1H,,f 6.9,4.8IJ2,C2-H);4.06(t'2H,J 6.8H:2,
cHr-orc); 4.32 (dd, !H, J 7.7,4.8IFr2, C4-H);4.52 (dd,
lH, Nl'-H); 5.59 (bs, lH, N3'-H).
r3C
LIJ, J
7.7,4.8H:2, C3-H); 5.21(bs,
NMR: 14.1,22.7,24.8,25.9,28.2,28.3,28.6,29.3,
29.4,2g.5,29.6,29.6,29.7,31.9,33.9,40.5,55.4,60.1,6L.9,64.6(alkyl),
163.4,L73.8
(carbonyl).
Biotin hexan-l-ol ester (160)
Recovered 110mg (BIVo) as a colourless amorphous solid mp
ll9-120'. HRMS Calculated
for c,uÌlrNrors: 328.I82I. Found: 328.1827. MS: 328 (M*, 19), 268 (I4),227 (I9),166
(39), 105 (100). lH NMR: 0.89 (1, 3lH^,J 6.3IcL2, CfL-CfL); 1.29-1.71(m, L4H, methylene
protons); 2.33 (t,2]H^, J 7 .Olfrz, Cf!-COr); 2.73
J
E
12.8,
4.8]
cr!-orc);
12,
C5-Ha); 3.15
(dt,lH,
,r
7
.4,
(d,lH, ,fB* I2.8Hz, C5-Hb); 29I (dd, lIJ,
4.8Il2, C2-H); 4'05 (t,ZIJ, J 6'7 Hz'
4.30 (dd,lH, -r 7.7,4.8IFr2, C4-IH); 4.51 (dd,LIJ,17.7,4.8}J2, C3-H); 5.38 (ås,
lH, Nl'-H); 5.78 (bs, lH, N3'-H).
t3C
NMR: 13.9,22.5,24.8,25.5,28.2,28.4,28.6,31.4,
33.9, 40.5,55.4, 60.1, 61.9, &5 (alkyl), 163.8, 173.8 (carbonyl)'
Biotin propan-l-ol ester (161)
A mixture of biotin (a1) (100m g,4.1mmol), propan-l-ol (3ml) andp-TsOH (7'8mg' 0'04
mmol,0.1eq) was refluxed for 6 hours, at which time a homogeneous mixture was formed.
The reaction mixture was cooled to room temperature, crystallised unreacted biotin removed
by filtration, the solvent removed in vacuo and the residue purified by flash chromatography
Experimental 144
eluant MeOfVCf!Clr5:95 to give the title compound as colourless crystals mp 126-127" in
73mg(62Vo)yield. HRMS: CalculatedforC,rtlrNrOrS: 286.1351'. Found: 286-1362.
MS: 286 (M*,7), 227 (82),166 (84),97 (100). 'HNMR: 0.95 (e 3}j,J7.5}l2, CHr-Ctl);
1.43-L.7 8
(2, 8H, methylene protons); 2.34 (t, 2IJ, I
7
.3 }Jz, CI{r-COr-) ; 2.7 4 (d,
lH, J s^
l2.8Hz,C5-Hb); 2.93 (dd,lH, {s," 12.8,4.9 Hz, CS-Ha);3.17 (dt,l}I,,f 8.0, 4.7 IJz,C2-H);
4.O4
(t,2H, .I 6.8 IFrz, Ct!-OrC); 4.33 (dd, lH,
"r 7
.6, 4.7 IJz, C4-H): 4.53 (dd, l}J, J 7 '6, 4'7
t'C NMR: 10.3,21.9,24.7,28.2,
Hz, C3-H);5.00 (bs, lH, Nl'-H); 5.34(bs,lH, N3'-H).
28.3, 33.9, 40.5, 55.4, 60. 1, 6 1.9, 65.9 (alkyl), 1 63.9,
L7
3.8 (carbonyl).
Biotin-undec-1O-yn-1-ol ester (162)
A mixture of biotin-NHS ester (106) (100mg,0.29 mmol), undec-10-yn-1-ol (59) (49mg'
0.29 mmol) and DMAP (3.6mg, 0.03 mmol, 0.1 eq) was stined in DMF (0.5m1) for 48 hours
at 50o. Silica gel (1g) was added and the solvent removed under vacuum (oil pump). The
residue was purified by flash chromatography eluant MeOIVCT!C126/94 to give the title
compoundas acolourless amorphous solidmp 84-86" ln52.6mg(46Vo) yield. HRMS
Calculated for
C,HroNPrS: 394.2290. Found: 394.2298. MS: 394 (M*, 6), 334 (18),227
(62), t66 (37),97 (100). IR (CDCL solution): 3476br m (N-H), 3304s (H-G), 2140w
(GC), lTl}brs (c=o). tH NMR:
H-G);
2.18 (dt, lljr, f 6.8,2.6 Hz,
I2.8Hz, C5-Hb); 2.92 (dd,lH, .f
1.25-1.77 (m, methylene protons); 1.94 (r, lH, J 2.6}12,
CF!-G); 2.29 (t,2IJ, J 7 -t Hz, Ct!-COr);
L2.8, 4.8 Hz, C5-Ha); 3.16
2.7
4 (d, lH, "r
(dt, l}I, J 7 .4, 4.7 Hz, C2-H);
4.05 (r, 2IH, f 6.6H2, CHr-OrC); 4.31 (dd,lH, J 7 .6, 4.7 Hz, C4-H); 4.51 (dd, tH, J 7 .6, 4'8
t'C NMR: 20.4,26-8,27.9,30.3,
Hz, C3-H);5.43 (bs, 1H, Nl'-H); 5.81 (ås, lH, N3'-H).
30.4, 30.5, 30.6,30.7 ,3I.0,31.2,3!.3,35.9, 42.5,
(alkynyl), 165.5,
17
5.7 (carbonyl).
57
.4, 62.I, 64.0,66.6 (alkyl),70.1,86.7
References I45
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